CN101678125A - Novel formulations for delivery of antiviral peptide therapeutics - Google Patents

Abstract

The invention provides compositions and methods for their administration as therapeutic agents. In particular, the invention provides compositions and their use for the administration of antiviral peptide therapeutics.

Description

The novel formulation that is used for the delivery of antiviral peptide therapeutic agent

Technical field

The invention provides compositions and with its method of using as therapeutic agent.Especially, the invention provides compositions and using for example purposes in the antiviral peptide therapeutic agent of bioactive molecule.

Background technology

The delivery of peptides system

Peptide product has purposes widely as being used for prevention and treatment treatment of diseases agent and/or preventive.Such peptide product belongs to various classifications, for example, for instance, hormone, enzyme and immunomodulator (for example antibody, serum albumin and cytokine).

For show the appropriate biology and the peptide of therapeutic effect in patient's body, they must be present in action site in the body with suitable concentration.More specifically, the pharmacokinetics of any specific compound (comprising any particular peptide) depends on this chemical compound bioavailability, distribution and clearance rate in vivo.Yet, the chemical property of peptide and characteristic, for example size, complexity, conformation require and solubility properties, are easy to make the pharmacokinetic property ideal of the pharmacokinetic property of peptide not as other chemical compound.

Therefore, this area has extensive work to be devoted to develop for example method of peptide of administering therapeutic agent, with bioavailability and the half-life that increases this therapeutic agent.Though make progress to some extent in this respect, this area still needs to can be used for sending with ideal pharmacokinetic property the other compositions of peptide therapeutics.Compositions provided by the invention and method have solved these demands.

Summary of the invention

The invention provides compositions, said composition for example can be used to use bioactive molecule to the patient.Particularly, the invention provides the compositions that comprises solvent, Binder Materials and bioactive molecule (for example antiviral peptide).Without being limited by theory, embodiment provided by the invention is at least in part based on unexpected discovery, promptly can in compositions when individuality is used the ideal pharmacokinetic property of demonstration, in compositions, improve the percentage by weight of the bioactive molecule that is comprised.

In one embodiment, after being applied to the patient, the biomolecule plasma concentration that compositions provided by the invention produces (for example in 8,12,16,20,24,28,32,36 or 48 hours) rapidly reaches C _MaxAnd in 5,7,10,14,17,21 or 28 days or longer time, provide the relative constant plasma concentration of this biomolecule.In specific embodiments, the desirable pharmacokinetic property of compositions provided by the invention is lower C _Max, longer t _MaxLonger t _0.01Or t _0.1

Compositions provided by the invention for example can be used for using the compositions that comprises some antiviral peptide, described antiviral peptide is called as T20 (SEQ ID NO:2), T1249 (SEQ ID NO:57), T897 (SEQ IDNO:58), T2635 (SEQ ID NO:5), T999 (SEQ ID NO:59) and T1144 (SEQ ID NO:9), the perhaps combination of two or more above-mentioned peptides, and T20 (SEQ ID NO:2), T1249 (SEQ IDNO:57), T897 (SEQ ID NO:58), T2635 (SEQ ID NO:5), the derivant of T999 (SEQ ID NO:59) and each peptide of T1144 (SEQ ID NO:9).

In one embodiment, described compositions comprises solvent, forms the Binder Materials and at least a bioactive molecule of substrate by solvent-subcutaneous fluid exchange, for example antiviral peptide, for example derivant of T20 (SEQID NO:2), T1249 (SEQ ID NO:57), T897 (SEQ ID NO:58), T2635 (SEQ ID NO:5), T999 (SEQ ID NO:59) and T1144 (SEQ ID NO:9) or above-mentioned peptide.

In another embodiment, these compositionss further comprise at least a extra composition, for example pharmaceutical acceptable carrier, macromole or its combination.In another embodiment, these compositionss can further comprise antiviral agent except the above antiviral peptide of enumerating.

The present invention further provides and used method for compositions provided by the invention.In one embodiment, described compositions is used as for example part of antiviral therapy scheme of therapeutic scheme.In certain embodiments, such therapeutic scheme is passable, for example, is used to treat HIV and infects, and for example HIV-1 infects.

In one embodiment, the invention provides and use compositions provided by the invention to suppress the method that HIV transmits to target cell, described method comprises to the patient uses a certain amount of compositions provided by the invention, so that described target cell is contacted by the activating agent of viral infection (for example antiviral peptide and/or another antiviral agent) with a certain amount of effective inhibition cell.

The method that the present invention also provides treatment HIV to infect (in one embodiment, HIV-1 infects), described method comprise the compositions provided by the invention of the effective dose of patient's administering therapeutic HIV infection of infecting to HIV.

The present invention further provides the preparation of compositions of using the bioactive molecule (for example antiviral peptide) that comprises effective dose and be used for the treatment of the medicine that HIV the infects method of (for example be used to suppress method that HIV transmits, suppress method and/or the treatment that HIV merges or suppress the method that HIV infects).

In conjunction with the accompanying drawings, above-mentioned and other target of compositions provided by the invention and method, feature and benefit are incited somebody to action clear presenting in the following detailed description.

Description of drawings

Fig. 1 is the sketch map of HIV-1gp41, has shown seven amino acid repeat region 1 (HR1) and seven amino acid repeat region 2 (HR2) and other functional areas of gp41, and described HR2 comprises the leucine zipper sample motif INNYTSLI that knows.For illustrative purposes, with respect to HIV _IIIBThe gp160 of strain has shown exemplary natural acid sequence and amino acid sites numbering corresponding to HR1 and HR2.

The unrestricted purpose for explanation, Fig. 2 has shown the comparison of the natural acid sequence that comprises in the HR2 zone of the HIV-1 gp41 that is determined by a plurality of laboratory strains and clinical separation strain, wherein, shown some variation in the aminoacid sequence (for example polymorphism) with the coded representation of single-letter aminoacid.Be the example purpose, with the separated strain sequence of aminoacid sequence INNYTSLI comparison in the separated strain sequence of the top corresponding to HR2 leucine zipper sample motif.

Fig. 3 is for showing the synthetic sketch map of HIV fusion inhibitor peptides SEQ ID NO:9, and described HIV fusion inhibitor peptides has the aminoacid sequence of SEQ ID NO:9, and described synthetic use relates to the fragment condensation of 3 kinds of fragments of peptides assemblings.

Fig. 4 is for showing the synthetic sketch map of HIV fusion inhibitor peptides SEQ ID NO:9, and described HIV fusion inhibitor peptides has the aminoacid sequence of SEQ ID NO:9, and described synthetic use relates to the fragment condensation of 2 kinds of fragments of peptides assemblings.

Fig. 5 has shown the intravital T1144 plasma concentration of macaque figure in after using T1144 by following compositions 432 hours: use ZnSO ₄The T1144 of solution precipitation (89% peptide) is at 74: 11: 15 SAIB: PLA3L: the 100mg/g suspension that forms among the NMP, 1000 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (60% peptide) was at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--■--).

Fig. 6 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: use ZnSO ₄The T1144 of solution precipitation (89% peptide) is at 74: 11: 15 SAIB: PLA3L: the 100mg/g suspension that forms among the NMP, 400 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (60% peptide) was at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--■--).

Fig. 7 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Use ZnSO ₄The T1144 of solution precipitation (89% peptide, 2% zinc) is at 80: 0: 20 SAIB: PLA3M: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (89% peptide, 2% zinc) is at 75: 5: 20 SAIB: PLA3M: the 50mg/g suspension that forms among the NMP, 400 μ l (--■--); Use ZnSO ₄The T1144 of solution precipitation (89% peptide, 2% zinc) is at 65: 15: 20 SAIB: PLA3M: the 50mg/g suspension that forms among the NMP, 400 μ l (--▲--).

Fig. 8 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) is at 85: 0: 15 SAIB: PLA3M: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) is at 74: 11: 15 SAIB: PLA3M: the 50mg/g suspension that forms among the NMP, 400 μ l (--■--).

Fig. 9 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) is at 70: 10: 20 SAIB: PLA3M: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) is at 75: 5: 20 SAIB: PLA3M: the 50mg/g suspension that forms among the NMP, 400 μ l (--■--).

Figure 10 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) is at 70: 10: 20 SAIB: PLA3M: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) is at 74: 11: 15 SAIB: PLA3M: the 50mg/g suspension that forms among the NMP, 400 μ l (--■--).

Figure 11 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) is at 75: 5: 20 SAIB: PLA3M: the 50mg/g suspension that forms in the glyceryl triacetate, 400 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) is at 75: 5: 20 SAIB: PLA3M: the 50mg/g suspension that forms in the benzyl benzoate, 400 μ l (--■--); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) is at 75: 5: 20 SAIB: PLA3M: the 50mg/g suspension that forms among the NMP, 400 μ l (--▲--).

Figure 12 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) is at 75: 5: 20 SAIB: PLA3M: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) is at 75: 5: 20 SAIB: PLA3M: the 75mg/g suspension that forms among the NMP, 400 μ l (--■--); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) is at 75: 5: 20 SAIB: PLA3M: the 100mg/g suspension that forms among the NMP, 400 μ l (--▲--).

Figure 13 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Use ZnSO ₄The T1144 of solution precipitation (88% peptide, 2% zinc) is at 77: 15: 8 SAIB: NMP: the 50mg/g suspension that forms in the ethanol, 400 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (88% peptide, 2% zinc) is at 77: 15: 8 SAIB: NMP: the 100mg/g suspension that forms in the ethanol, 200 μ l (--■--); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) is at 74: 11: 15 SAIB: PLA3M: the 100mg/g suspension that forms among the NMP, 400 μ l (--◇--); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) is at 74: 11: 15 SAIB: PLA3M: the 100mg/g suspension that forms among the NMP, 200 μ l (----).

Figure 14 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Use ZnSO ₄The T1144 of solution precipitation (89% peptide, 2% zinc) is at 75: 5: 20 SAIB: PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (89% peptide, 2% zinc) is at 75: 5: 20 SAIB: PLA3M: the 50mg/g suspension that forms among the NMP, 400 μ l (--■--).

Figure 15 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Use ZnSO ₄The T1144 of solution precipitation (73%) is at 75: 5: 20 SAIB: PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (89%) is at 75: 5: 20 SAIB: PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--■--); Use ZnSO ₄Solution precipitation (73%) and at ZnSO ₄The T1144 of slurrying in the solution (70%) is at 75: 5: 20 SAIB: PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--▲--); Spray drying (89%) and at ZnSO ₄The T1144 of slurrying in the solution (66%) was at 75: 5: 20 SAIB: the 50mg/g suspension that forms among the PLA3L:NMP, 400 μ l (--zero--); Use ZnSO ₄Solution precipitation (88%) and at ZnSO ₄The T1144 of slurrying in the solution (71%) is at 75: 5: 20 SAIB: PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--Ж--); Spray drying (89%) and at ZnSO ₄The T1144 of (65%) suspend in the solution at 75: 5: 20 SAIB: PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--x--).

Figure 16 has shown the intravital T1144 plasma concentration of monkey figure in after using T1144 by following compositions 432 hours: 3.5mg/mL T1144 solution, 800 μ l (-); Use ZnSO ₄The T1144 of solution precipitation (89%) is at 75: 5: 20 SAIB: PLA3M: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (89%) is at 74: 11: 15 SAIB: PLA3L: the 100mg/g suspension that forms among the NMP, 400 μ l (--■--); Use ZnSO ₄The T1144 of solution precipitation (89%) is at 74: 11: 15 SAIB: PLA3L: the 100mg/g suspension that forms among the NMP, 1000 μ l (--▲--).

Figure 17 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Use ZnSO ₄The T1144 of solution precipitation (89% peptide, 2% zinc) was at 40: 60 PLGA1A: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (89% peptide, 2% zinc) was at 60: 40 PLGA1A: the 50mg/g suspension that forms among the NMP, 400 μ l (--■--); Use ZnSO ₄The T1144 of solution precipitation (88% peptide, 2% zinc) was at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--▲--).

Figure 18 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) was at 50: 50 PLGA1A: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) was at 40: 60 PLGA1A: the 50mg/g suspension that forms in the glyceryl triacetate, 400 μ l (--■--); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) was at 40: 60 PLGA3A: the 50mg/g suspension that forms among the NMP, 400 μ l (--▲--); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) was at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--zero--).

Figure 19 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) was at 50: 50 PLGA1A: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); Use ZnSO ₄The T1144 of solution precipitation (73% peptide, 2% zinc) was at 50: 50 PLGA1A: the 100mg/g suspension that forms among the NMP, 400 μ l (--■--); Use ZnSO ₄The T1144 of solution precipitation (91% peptide, 2% zinc) was at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--◇--); Use ZnSO ₄The T1144 of solution precipitation (91% peptide, 2% zinc) was at 40: 60 PLA3L: the 100mg/g suspension that forms among the NMP, 400 μ 1 (----); Use ZnSO ₄The T1144 of solution precipitation (90% peptide, 2% zinc) was at 40: 60 PLA3L: the 200mg/g suspension that forms among the NMP, 400 μ l (--△--).

Figure 20 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); The T1144 of spray drying preparation was at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); The sedimentary T1144 of pH (88% peptide) was at 40: 60 PLA3L in methanol: the 50mg/g suspension that forms among the NMP, 400 μ l (--■--).

Figure 21 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Use ZnSO ₄The T1144 of solution precipitation (60%) was at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); Washed T1144 (94%) was at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--■--); Use ZnSO ₄The T1144 of solution precipitation (88%) was at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--▲--); The T1144 that is settled out from methanol (91%) was at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--zero--).

Figure 22 has shown the intravital T1144 plasma concentration of rat figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Use CaCl ₂The T1144 of solution precipitation (29%) was at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); Use CaCl ₂The T1144 of solution precipitation (53%) was at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--■--); Use FeSO ₄The T1144 of solution precipitation (88%) was at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--▲--); Use FeSO ₄The T1144 of solution precipitation (91%) was at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--zero--).

Figure 23 has shown the intravital T1144 plasma concentration of monkey figure in after using T1144 by following compositions 432 hours: 3.5mg/mL T1144 solution, 800 μ l (-); With the PLGA1A of the sedimentary T1144 of zinc solution (89%) at 40: 60: the 50mg/g suspension that forms among the NMP, 400 μ l (--◆--); With the sedimentary T1144 of zinc solution (60% peptide) at 40: 60 PLA3L: the 50mg/g suspension that forms among the NMP, 400 μ l (--■--).

Figure 24 has shown the intravital T1144 plasma concentration of monkey figure in after using T1144 by following compositions 168 hours: 3mg/mL T1144 solution, 1200 μ l (-); Precipitation H was at 40: 60 PLA3L: the suspension among the NMP, 400 μ l (----); Precipitation J was at 40: 60 PLA3L: the suspension among the NMP, 400 μ l (--◇--); Precipitation M was at 40: 60 PLA3L: the suspension among the NMP, 400 μ l (--◆--); Precipitation N was at 40: 60 PLA3L: the suspension among the NMP, 400 μ l (--■--).

Describe in detail

Definition

When being used for specification and claims in this article, term " patient " refers to mammal, for example people. In specific embodiments, " patient " is the mammal that needs disease disclosed by the invention or illness (for example HIV or AIDS) treatment, for example people.

When being used in this article specification and claims, term " target cell " refers to the cell that can be infected by HIV. In one embodiment, described cell behaviour cell; In another embodiment, described cell is people's cell that can be infected by HIV by the process that comprises the film fusion. In another embodiment, described cell is present in the patient for example in people's patient body.

When being used for specification and claims in this article, term " composition " refers to comprise the preparation of solvent, Binder Materials and bioactive molecule (for example antiviral peptide). This paper describes exemplary composition. Composition provided herein can for example be used as medicine or for the preparation of medicine.

When being used in this article specification and claims, term " solvent " refer to can be miscible with water liquid. In one embodiment, described solvent be can with the miscible liquid of water and with cosolvent for example NMP be used in combination. Solvent can be used to, and for example, fully dilutes Binder Materials and is injected in the patient body allowing. The invention describes exemplary solvent, described exemplary solvent is well known to those skilled in the art.

When being used for specification and claims in this article, term " Binder Materials " refer to can be miscible with solvent material, described material forms matrix by solvent-subcutaneous fluid exchange (being solvent-subcutaneous patient's fluid exchange) in being present in the composition that comprises solvent and Binder Materials the time. The invention describes exemplary Binder Materials, described exemplary Binder Materials is well known to those skilled in the art.

When being used in this article specification and claims, term " matrix " but refer to the form of the biodegradable or bioerosion that Binder Materials presents after solvent-subcutaneous fluid exchange. In one embodiment, described matrix is viscosity (namely anti-shearing) matrix. In another embodiment, described matrix is gel.

When being used for specification and claims in this article, term " carrier " refers to can be used for send to the patient fluent material that comprises solvent and Binder Materials of bioactive molecule (for example peptide, for example antiviral peptide). Carrier can be stored with aqueous state.

When being used in this article specification and claims, term " pharmaceutically acceptable carrier " refers to be added in the active component (for example Fuzeon) and does not significantly change the mounting medium of the BA of described active component. Pharmaceutically acceptable carrier includes but not limited to one or more following materials: water, buffered water, salt solution, 0.3% glycine, aqueous alcoholic, isotonic water solution; And can further comprise one or more materials, for example glycerine, oils, salt (for example sodium salt, sylvite, magnesium salts and ammonium salt), phosphate, carbonic ester, aliphatic acid, carbohydrate (for example sweet mellow wine), polysaccharide, polymer, excipient and anticorrisive agent and/or stabilizing agent (be used for extending the shelf life or for composition production and selling institute essential and suitable). In one embodiment, pharmaceutical acceptable carrier is suitable for interior, the subcutaneous or parenteral administration of muscle.

Unless otherwise specifically indicated, when being used for this specification and claims and relating to Fuzeon, term " comprises isoleucine or leucic amino acid " and refers to respectively isoleucine or leucine, or described their corresponding naturally occurring amino acid (such as L-amino acid), the non-natural amino acid (such as D-amino acid), isomeric forms (such as nor-leucine, alloisoleucine etc.) or the derivative form (for example Terleu) that exist. A kind of form of the amino acid of isoleucine or leucine can be used for getting rid of described amino acid whose other form. Fuzeon as herein described also can in its amino acid sequence, comprise one or more polymorphisms of being derived from HIV gp41HR2 regional sequence (referring to, Fig. 2 for example), except comprising, the one or more sites in the amino acid sequence of the present invention's instruction comprise isoleucine or leucic amino acid.

Term " HIV " refers to HIV (human immunodeficiency virus), in one embodiment, refers to HIV-1.

Term " isolating " is when being used for bioactive molecule (for example antiviral peptide such as HIV fusion inhibitor peptides or fragments of peptides), refer to the composition that described bioactive molecule does not contain does not substantially become the integrally-built part of this biomolecule self, for example, when use biology, biochemistry or chemical method are synthesized, prepared or modify, do not contain precursor or other chemical drugs substantially.In certain embodiments, isolating bioactive molecule purity surpasses about 75%, 80%, 85%, 90%, 95%, 97%, 99% or 99.9% weight.

When being used for the HIV fusion inhibitor peptides, term " 1 to 3 aminoacid is replaced " refers to that the HIV fusion inhibitor peptides still has the aminoacid sequence of arbitrary sequence among the SEQ ID NO:9-15, still compares to have with arbitrary sequence among the SEQ ID NO:9-15 to be no less than 1 and no more than 3 aminoacid differences; Also have simultaneously (a) more than one leucine zipper sample motif and except forming the needed leucine of leucine zipper sample motif at least one extra leucine in (promptly except that 1 or 8 sites of leucine zipper sample motif), or (b) 3 to 5 leucine zipper sample motifs; And has the antiviral activity (activity that suppresses the fusion of HIV mediation) of anti-HIV.In this regard, the amino acid difference dystopy of HIV fusion inhibitor peptides with replacement (when comparing with SEQ ID NO:9-15) is (referring to, the present invention's diagram (I) and (II) for example) in the aminoacid sequence site except that HIV fusion inhibitor peptides of the present invention is denoted as leucine and/or isoleucine residue.Describedly be no less than 1 and no more than 3 aminoacid differences and include, but not limited to conserved amino acid and replace and (known in the artly comprise and be replaced the aminoacid that aminoacid has essentially identical electric charge, size, hydrophilic and/or armaticity and replace; Example comprises, but be not limited to, glycine-alanine-valine, tryptophan-tyrosine, aspartic acid-glutamic acid, arginine-lysine, agedoite-glutamine and serine-threonine) and/or polymorphism is (for example, as shown in Figure 2, or as clinical separation strain that find at laboratory, multiple differentiation branch and/or HIV-1).For example, for SEQ ID NO:11,12 or 13, the HIV fusion inhibitor peptides has 1 to 3 aminoacid difference, the site beyond the amino acid residue 10,17,24,31 of described amino acid difference dystopy arbitrary sequence in SEQ ID NO:11,12 or 13 and 38.For example, for SEQ ID NO:9 and SEQ ID NO:14, the HIV fusion inhibitor peptides has 1 to 3 aminoacid difference, the site of described amino acid difference dystopy beyond the amino acid residue 10,17,21,24 and 38 of SEQ ID NO:9 or SEQ ID NO:14.For example, for SEQ ID NO:10 or SEQ ID NO:15, the HIV fusion inhibitor peptides has 1 to 3 aminoacid difference, the site of described amino acid difference dystopy beyond the amino acid residue 10,17,21,31 and 38 of SEQ ID NO:10 or SEQ ID NO:15.The illustrative example of described embodiment includes but not limited to the aminoacid sequence of SEQ ID NO:16, wherein may be that the amino acid difference ectopic sites that 1 to 3 aminoacid is replaced indicates (represent any aminoacid natural or that non-natural exists, promptly can be used for this amino acid sites more than a kind of possible aminoacid) with Xaa.Similarly, can produce one or more conserved amino acids and replace, for example replace lysine, replace arginine, replace glutamic acid or replace aspartic acid with glutamic acid with aspartic acid with lysine or histidine with arginine or histidine.Be illustrative purpose, amino acid sites 10,17,21,24,31 and 38 usefulness underscores indicate.Equally for SEQ ID NO:9-15, should notice that " Zaa " is used to indicate in SEQ ID NO:16 and may be the aminoacid of leucine or isoleucine; And Baa is used for sign and is preferably leucine, isoleucine, but may be the aminoacid of Xaa, but at least one Baa is leucine or isoleucine.

SEQ?ID?NO：16：

XaaXaaXaaEAXaaDRA ZaaAEXaaAAR ZaaEAZaa ZaaRA BaaXaaEXaaXaaEK BaaEAAZaaRE Zaa

HIV fusion inhibitor peptides of the present invention is also passable, for example, comprise HIV gp41 HR2 zone deutero-peptide (passing through sequence alignment) corresponding to SEQ ID NO:5, described peptide is present in laboratory strains, differentiation branch or the clinical separation strain of HIV-1, listed laboratory strain and clinical separation strain among Fig. 2 for example is as long as described HIV fusion inhibitor peptides satisfies the aminoacid requirement of SEQ ID NO:16.In one embodiment, these HIV fusion inhibitor peptides are compared with arbitrary sequence among the SEQ ID NO:9-15, demonstrate 1 to 3 aminoacid and replace.In one embodiment, described HIV fusion inhibitor peptides further comprises N-terminal blocking groups or C-terminal blocking groups, or above-mentioned both; And described end group can include, but are not limited to: the amino or the acetyl group that are positioned at N-terminal; Be positioned at the carboxyl or the amide groups of C-terminal.

HIV fusion inhibitor peptides of the present invention also can comprise the peptide of the variant aminoacid sequence that demonstrates the disclosed any peptide of U.S. Pat 2006/0247416 (by reference its full content being incorporated herein), as long as described HIV fusion inhibitor peptides satisfies the aminoacid requirement of SEQ ID NO:16.In one embodiment, this type of HIV fusion inhibitor peptides is compared with arbitrary sequence among the SEQ ID NO:9-15, demonstrates 1 to 3 aminoacid and replaces.In preferred embodiments, the length of described HIV fusion inhibitor peptides is 14 to 60 amino acid residues.In one embodiment, described HIV fusion inhibitor peptides further comprises N-terminal blocking groups or C-terminal blocking groups, or above-mentioned both; And described end group can include but not limited to: the amino or the acetyl group that are positioned at N-terminal; Be positioned at the carboxyl or the amide groups of C-terminal.

When being used for description and claims in this article, term " reactive functional groups " refers to form with another chemical group or chemical part the chemical group or the chemical part of chemical bond.About chemical group; those skilled in the art know; the group that reactive functional groups comprises includes but not limited to, maleimide, sulfydryl, carboxylic acid, hydrogen, phosphoryl, acyl group, hydroxyl, acetyl group, hydrophobic group, amine, amide groups, dansyl, sulfo group, butanimide, sulfydryl reactive group, amine reactive group, carboxyl-reactive group etc.A kind of reactive functional groups can be used for getting rid of another reactive functional groups.

When being used for description and claims in this article, term " joint " as the molecule bridge with the chemical compound of two different moleculars that are operably connected or part (for example refers to, the covalently bound reactive functional groups of first reactive functional groups of joint, and the covalently bound reactive functional groups of second reactive functional groups of this joint) to the HIV fusion inhibitor peptides to macromolecular carrier.In the preparation of the recombination fusion protein of the one or more copies that comprise the HIV fusion inhibitor peptides, joint can be an aminoacid.Perhaps, two different moleculars can be connected on the joint in the mode (for example by chemical coupling) of substep.Generally speaking, for joint, there is not the restriction of specific size or composition, as long as described joint can satisfy the purposes as the molecule bridge.Those skilled in the art know, and joint includes but not limited to, chemical chain, chemical compound (for example reagent), aminoacid etc.Joint can include but not limited to, same bifunctional linker, isodigeranyl functional connector, Biostatic joint, hydrolyzable joint and biodegradable joint, and other joint known in the art.Those skilled in the art know, thereby the isodigeranyl functional connector comprises and has the end that the first reactive functional groups specificity connects first molecule, thus with have the second reactive functional groups specificity and be connected the opposite end of second molecule.It will be apparent for a person skilled in the art that multiple single function, difunctional and poly functional reagent (for example Pierce Chemical Co., Rockford, the reagent described in the III catalogue) can be used as joint.According to for example treating connected molecule, the condition that connects and use the factors such as pharmacokinetic property of time expectation, described length of said joint and forming can change to optimize for example following character: the maintenance of biological activity and function, stability, to the resistance of certain chemicals and/or temperature parameter, in vivo to cracked sensitivity and enough stereo selectivity or sizes.

When being used for description and claims in this article, term " macromolecular carrier " (for example refers to connect, adds or merges, chemically, or by using the recombination method of genetic expression) to the molecule of one or more peptides of the present invention, compare with the described one or more peptides that do not have the described minute period of the day from 11 p.m. to 1 a.m thus, described molecule can give described one or more peptide one or more following character: the prolongation of stable, bioactive enhancing, plasma half-life (for example prolonging described one or more peptide persistency in vivo).Well known, these macromolecular carriers include but not limited to, serum albumin, polymer, carbohydrate and lipid-fatty acid conjugates.Typically the serum albumin as macromolecular carrier includes but not limited to, transferrins, albumin (preferred human albumin), immunoglobulin (preferred human IgG or its one or more chain) or hormone.Typically the polymer as macromolecular carrier includes but not limited to, polylysine or poly-(D-L-alanine)-poly-(L-lysine) or polyhydric alcohol.Polyhydric alcohol can comprise water-soluble poly (epoxyalkane) polymer, and can have straight or branched.Polymer can be branched chain polyol (PEG for example has many (for example 3 or more) bar chain, and every chain all can directly or by joint be connected to the HIV fusion inhibitor peptides); In one embodiment, polymer can be biodegradable and/or cracked in time branched chain polyol under the condition in vivo.Suitable polyhydric alcohol includes but not limited to, Polyethylene Glycol (PEG), polypropylene glycol (PPG) and PEG-PPG copolymer.In one embodiment, polyhydric alcohol comprises PEG, and the mean molecule quantity of described PEG is selected from from about 1000 dalton to about 20000 daltonian scopes.Spendable other type macromolecular carrier known in the art, described carrier have usually and are higher than 20000 daltonian molecular weight.

When being used for description and claims in this article, term " chemoproection group " or " CPG " refer to be used to block the chemical part that the reactive functional groups that contains amine groups and another reactive functional groups carry out chemical reaction.The technical staff in the synthetic field of peptide knows; the chemoproection group includes but not limited to; tBu (tert-butyl group); trt (trityl (trityl)); OtBu (tert-butoxy); Boc or t-Boc (tertbutyloxycarbonyl); Fmoc (9-fluorenylmethyloxycarbonyl); Aoc (uncle's penta oxygen carbonyl); TEOC (β-trimethyl carbethoxyl group); CLIMOC (2-chloro-1-indanyl methoxycarbonyl group); BIMOC (benzene-[f]-indenes-3-methoxycarbonyl group); PBF (2; 2; 4; 6; 7-pentamethyl Dihydrobenzofuranes-5-sulfonyl); 2-Cl-Z (benzyloxycarbonylchloride base); Alloc (allyloxycarbonyl); Cbz (benzyloxycarbonyl group); Adoc (Buddha's warrior attendant alkoxy carbonyl group); Mcb (methyl ring butoxy carbonyl); Bpoc (2-(right-xenyl) propyl group-2-oxygen carbonyl); Azoc (2-(right-the phenylazo phenyl) propyl group-2-oxygen carbonyl); Ddz (2; 2 dimethyl-3; 5-veratryl-oxygen carbonyl); MTf (4-methoxyl group-2; 3; 6-trimethoxyphenyl sulfonyl); PMC (2; 2; 5; 7; 8-pentamethyl chromane-6-sulfonyl); Tos (p-toluenesulfonyl); Hmb (2-hydroxyl-4-methoxybenzyl); Poc (2-phenylpropyl-2-oxygen carbonyl); (1-(4 for Dde; 4-dimethyl-2; 6-dioxo hexamethylene-1-subunit) ethyl); ivDde (1; (4; 4-dimethyl-2,6-dioxo-hexamethylene-1-subunit)-the 3-methyl butyl); benzyl; dansyl; right-nitrobenzyl ester etc.A kind of chemoproection group can be used for getting rid of another kind of chemoproection group.

When being used for description and claims in this article; term known in the art " goes protection " and refers to that wherein said molecule comprises aminoacid, fragments of peptides or HIV fusion inhibitor peptides from the process of the one or more chemoproection groups of molecule removal that contain one or more chemoproection groups.Usually, go the protection process to relate to by the molecule of one or more chemoproection radical protections and the chemical reagent reaction of removing described chemoproection group.For example, by the terminal α amino of the N-of chemoproection radical protection can with alkali reaction to remove alkali labile chemoproection group (for example Fmoc etc.).Chemoproection group (for example Boc, TEOC, Aoc, Adoc, Bopc, Ddz, Cbz etc.) is removed with acid.The group of other chemoproection group, especially derived from carboxylic acid, usable acid or alkali are removed.

When being used for description and claims in this article, term " derivant " thus refer to by replacing the chemical compound that one or more atoms are produced by parent compound with another atom or another group atom, when wherein said chemical compound is antiviral compound, preferably keep all of parent compound or all antiviral activities substantially.

Term " first ", " second ", " the 3rd " etc. can be used in this article: (a) show order; Or (b) distinguish the reactive functional groups of molecule or molecule; Or (c), combination (a) and (b).But term " first ", " second ", " the 3rd " etc. should not be interpreted as restriction the present invention in addition.

When relating to HIV fusion inhibitor peptides of the present invention, term " fragments of peptides " and " intermediate " synonym in this article use, and when being used for description and claims in this article, refer to comprise the peptide that length is no less than the aminoacid sequence of about 5 aminoacid and no more than about 30 amino acid residues, and comprise at least a portion (successive aminoacid) of the aminoacid sequence of described HIV fusion inhibitor peptides.The illustrative example of fragments of peptides that can be used for preparing SEQID NO:9 and 10 is referring to embodiment of the invention 4-7, and table 4,5,7 and 8.In addition, when synthetic peptide fragment in preferred embodiments (individually or be combined into a group) when between amino acid residue, forming peptide bond thus to form the HIV fusion inhibitor peptides, it will be apparent for a person skilled in the art that and to use reaction well known to those skilled in the art to form non-peptide bond (for example imido grpup, ester, hydrazides, azo, semicarbazides etc.).

When being used for description and claims in this article, the total amount of bioactive molecule (for example antiviral peptide) in the systemic available compositions that term " pharmacokinetic property " refers to change along with the time.Can determine pharmacokinetic property by using the back time dependent whole body total concentration of mensuration described bioactive molecule (for example antiviral peptide) in vivo.For example, area (AUC), biological half-life and/or clearance rate are expressed under pharmacokinetic property usable concentration-time graph.AUC is the integrated metric of whole body bioactive molecule concentration in time, and unit is quality-time/volume.Use after the potion bioactive molecule, the AUC of the time point that self administration of medication time point non-activity composition to the body retains is the tolerance that individuality is exposed to described bioactive molecule (and/or metabolite of described bioactive molecule).Compare when having following one or more features when the bioactive molecule that comprises in the preparation beyond bioactive molecule that compositions comprised and the preparation of the present invention, described compositions has " improvement " or " raising " pharmacokinetic property: (a) longer (" raising ") biological (the terminal elimination) half-life (" t ¹/ ₂"), (b) reduction of biological (whole body) clearance rate (Cl), (c) longer sustained releasing property, (d) percentage by weight that in described compositions, improves (for example about 10% or higher), (e) reduce or lower C _Max, (f) longer t _MaxAnd (g) longer t _0.01Or t _0.1In one embodiment, the pharmacokinetics of improvement means with respect to being reduced than preparation bioactive molecule clearance rate, for example typically reduces about 2 times to about 10 times.In another embodiment, the pharmacokinetics of improvement means that with respect to by than preparation, biological half-life prolongs about 10% to about 60%.The pharmacokinetics of improving can comprise simultaneously that also clearance rate reduces and biological half-life prolongs.In one embodiment, ideal pharmacokinetic property comprises that rapidly (for example in 8,12,16,20,24,28,32,36 or 48 hours) reach C _Max, keep relative constant plasma concentration 5,7,10,14,17,21 or 28 days or longer time then.In specific embodiments, the desirable pharmacokinetic property of compositions provided by the invention is lower C _Max, longer t _MaxLonger t _0.01Or t _0.1Be used to calculate area under plasma concentration-time graph (AUC), whole body total body clearance (Cl) and the terminal half-life (t of elimination ¹/ ₂) formula in the embodiment of the invention 1, describe in detail.

When being used for description and claims in this article, as the standard terminology that is used for representing wherein to have dissolved one or more solid aqueous solutions in this area " solution " refer to the present invention that describe in detail with concentration and temperature practical application condition injectable drug formulation art standard under, comprised for example aqueous solution of HIV fusion inhibitor peptides of the bioactive molecule that is dissolved in wherein.Several different methods known in the art is used to distinguish solution morphology and suspension form, for example checks that vision clarity (solution transparent and suspension muddiness), light penetrate etc." dissolubility " by be present in the waterborne liquid solution bioactive molecule for example the amount of HIV fusion inhibitor peptides (for example percentage by weight) determine that described solution does not have visible precipitation, and gelling does not take place in the waterborne liquid that contains described bioactive molecule.

When being used for description and claims in this article, term " continues release " and refer to that after using bioactive molecule continuously discharges in particular time interval.

When being used for description and claims in this article, term " effective dose " refers to reach the amount of bioactive molecule of the desired result of ad hoc approach.In one embodiment, the effective dose of biomolecule can be to be enough to the amount that (combining by himself and/or with the multiple dose scheme) reduces HIV virus load in (when for example not existing with respect to described bioactive molecule) patient's body.In another embodiment, the effective dose of biomolecule can be to be enough to suppress the infection of (when for example not existing with respect to described bioactive molecule) HIV pair cell or to suppress the amount that virus enters target cell.These inhibition can use methods known in the art to measure.In another embodiment, the effective dose of biomolecule can be to be enough to improve HIV to infect related indication amount.Those skilled in the art can use data in the external and body of routine well known to those skilled in the art to determine the effective dose of biomolecule.

When relating to the HIV infection, the expression that is equal on term " treatment " or " therapy " or its grammer is used interchangeably, and when being used for description, refer to comprise with claims bioactive molecule for example the compositions of HIV fusion inhibitor peptides can be used to influence with HIV and infect one or more relevant processes, or influence is as the one or more parameters or the terminal point of the curative effect index of determining this type of treatment or therapy (for example " treatment application ").For example, bioactive molecule can be used for suppressing the transmission of following one or more processes: HIV to target cell; The fusion of HIV and target cell (" HIV fusion "); Virus enters (process that HIV or its hereditary material enter target cell in course of infection); And syncytium forms (for example fusion of HIV infection cell and target cell).For the effect of assessment HIV treatment of infection or therapy Chinese medicine, it is the main terminal point of using always that virus suppresses (being determined by the method that is used for measuring body fluid or organizing virus load known in the art), the CD4 in the blood circulation ⁺Increasing of cell quantity is the secondary endpoints of using always; Above-mentioned both suppresses the effect surveyed that HIV transmits to target cell.Therefore, comprise bioactive molecule for example the compositions of HIV fusion inhibitor peptides can be used for influence treatment and use, comprise that virus suppresses and/or circulation CD4 ⁺The cell relative populations increases.

Compositions

The invention provides the compositions that can be used for using bioactive molecule to the patient.Particularly, the invention provides the compositions that comprises solvent, Binder Materials and bioactive molecule (for example antiviral peptide).Without being limited by theory, embodiment provided by the invention to small part based on unexpected discovery, can in compositions, improve the percentage by weight of the bioactive molecule that is comprised and after being applied to the patient, demonstrate ideal sustained releasing property.Compositions of the present invention can constitute the gel combination that original position forms, because described compositions comprises substrate after solvent-subcutaneous fluid exchange being applied to the patient and taking place.

In one embodiment, when being applied to the patient, the biomolecule plasma concentration that compositions provided by the invention produces (for example in 8,12,16,20,24,28,32,36 or 48 hours) rapidly reaches C _MaxAnd in 5,7,10,14,17,21 or 28 days or longer time, provide the relative constant plasma concentration of described biomolecule.In specific embodiments, the desirable pharmacokinetic property of compositions provided by the invention is lower C _Max, longer t _MaxLonger t _0.01Or t _0.1

Compositions provided by the invention can be used for, for example, use the compositions that comprises some antiviral peptide, described antiviral peptide is known as T20 (SEQ ID NO:2), T1249 (SEQ ID NO:57), T897 (SEQ IDNO:58), T2635 (SEQ ID NO:5), T999 (SEQ ID NO:59) and T1144 (SEQ ID NO:9), two or more combination in the perhaps above-mentioned peptide, and T20 (SEQ ID NO:2), T1249 (SEQ IDNO:57), T897 (SEQ ID NO:58), T2635 (SEQ ID NO:5), the derivant of T999 (SEQ ID NO:59) and each peptide of T1144 (SEQ ID NO:9).

In one embodiment, described compositions comprises solvent, forms the Binder Materials and at least a bioactive molecule of substrate by solvent-subcutaneous fluid exchange, for example antiviral peptide, for example derivant of T20 (SEQID NO:2), T1249 (SEQ ID NO:57), T897 (SEQ ID NO:58), T2635 (SEQ ID NO:5), T999 (SEQ ID NO:59) and T1144 (SEQ ID NO:9) or above-mentioned peptide.Described compositions is passable, for example, is that solution or suspension exist using previous crops.Described solution or suspension can be aqueous or comprise organic solvent.Behind the liquid in being exposed to patient's subcutaneous space, but described Binder Materials can form biodegradable or the substrate of bioerosion at least.Therefore, in one embodiment, described compositions can be applied to liquid form this patient who needs, and for example, passes through subcutaneous injection.The substrate that is produced is passable, for example, and as the lasting release matrix of described bioactive molecule.

Compositions provided by the invention comprises at least a Binder Materials usually, the amount of described Binder Materials (for example, about 50-1000mg/g, 100-900mg/g, 150-900mg/g, 200-900mg/g, 250-900mg/g, 500-900mg/g, 750-900mg/g or 750-1000mg/g) is enough to form substrate after being applied to the patient.In one embodiment, the method for determining Binder Materials proper content (mg/g of unit) is to add the carrier Binder Materials from the carrier ratio to form, and multiply by 10 again.In one embodiment, described Binder Materials is lactide or co-glycolide polymers.In specific embodiments, described Binder Materials is Sucrose acetoisobutyrate (SAIB), polylactide (PLA, for example PLA3L, PLA3M or PLA-PEG) or polylactide-co-glycolide copolymer (PLG, PLGA be PLGA1, PLGA-glucose or PLGA-PEG for example).Compositions provided by the invention also can comprise bioactive molecule, for example antiviral peptide.In one embodiment, compositions provided by the invention comprises the bioactive molecule of enough concentration, thus can be from being applied to the described bioactive molecule of substrate (for example mode to continue to discharge) the release effective dose that forms behind the patient.

Compositions provided by the invention can comprise the also bioactive molecule of application concentration at least 5% usually.Therefore, in one embodiment, the amount of the bioactive molecule (for example antiviral peptide) that comprises of compositions provided by the invention equal composition weight about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20% or higher.

In specific embodiments, after being applied to the patient, compositions provided by the invention forms substrate, and described substrate provides the initial burst of described bioactive molecule, continues to discharge described bioactive molecule then at least in 5,7,10 or 14 days after described compositions is used.In certain embodiments, be C in behind applying said compositions 2,4,6,8,10,12,14,16,18,20 or 24 hours by initial burst from the bioactive molecule that compositions provided by the invention provides to the patient that use _MaxAt least or be approximately 1 μ g/ml, 2 μ g/ml, 3 μ g/ml, 4 μ g/ml, 5 μ g/ml, 6 μ g/ml, 7 μ g/ml, 8 μ g/ml, 9 μ g/ml, 10 μ g/ml, 11 μ g/ml, 12 μ g/ml, 13 μ g/ml, 14 μ g/ml or 15 μ g/ml or higher.In certain embodiments, by the lasting release of using the bioactive molecule that compositions provided by the invention provides to the patient be behind applying said compositions at least 5,7,10,14,21,25 or 28 days or longer time more than or equal to 0.1 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, 1.25 μ g/ml, 1.5 μ g/ml or 2.0 μ g/ml or higher.

In another embodiment, the invention provides compositions, said composition forms substrate (for example original position) after being applied to the patient, and the C of the bioactive molecule (for example antiviral peptide) of at least 10 μ g/ml is provided in 12 hours of using _Max, continue then to discharge, make that blood plasma level is at least 1 μ g/ml at least 7 days.

In one embodiment, the compositions that comprises antiviral peptide provided by the invention further comprises at least a extra composition, for example pharmaceutical acceptable carrier, macromole or its combination.

In another embodiment, described compositions comprises one or more extra bioactive molecules.In one embodiment, described compositions can comprise the derivant of T20, T1249, T897, T2635, T999 or T1144 peptide or above-mentioned peptide.In another embodiment, this based composition can comprise or further comprise one or more other antiviral agent.Can be separately or include but not limited to as other antiviral agent that the part of combined therapy scheme is used for compositions, DP107 (T21) or any other antiviral polypeptide, for example United States Patent (USP) 6, antiviral polypeptide described in 541,020 B1 (by reference its full content being attached to herein).Other example exemplary, nonrestrictive therapeutic agent comprises for example cytokine of antiviral agent, for example rIFN α, rIFN β, rIFN γ; Reverse transcriptase inhibitors, include but not limited to Abacavir, AZT (zidovudine), ddC (zalcitabine), nevirapine, ddl (Didanosine), FTC (emtricitabine), (+) and (-) FTC, reverset, 3TC (lamivudine), GS 840, GW-1592, GW-8248, GW-5634, HBY097, dilazep Wei Ding, efavirenz, d4T (stavudine), FLT, TMC125, adefovirdipivoxil, Tai Luofuwei and alovudine; Protease inhibitor, include but not limited to amprenavir (amprenivir), CGP-73547, CGP-61755, DMP-450, indinavir, nelfinavir, PNU-140690, ritonavir, Saquinavir, Compound E, tipranavir (tipranovir), atazanavir, Lopinavir, ABT378, ABT538 and MK639; Virus mRNA adds medicated cap inhibitor, for example ribavirin; Amphotericin B as lipid binding molecule with HIV (human immunodeficiency virus)-resistant activity; Castanospermine (castanospermine) as glycoprotein processing inhibitor; Viral entry inhibitor, for example if fusion inhibitor (En Fuwei ground, T1249, other fusion inhibitor peptides, and micromolecule), SCH-D, UK-427857 (Pfizer), TNX-355 (Tanox Inc.), AMD-070 (AnorMED), Pro 140, Pro 542 (Progenies), FP-21399 (EMD Lexigen), BMS806, BMS-488043 (Bristol-Myers Squibb), Malawi are (UK-427857), ONO-4128, GW-873140, AMD-887, CMPD-167 and GSK-873,140 (GlaxoSmithKline); CXCR4 antagonist, for example AMD-070; Lipid and/or cholesterol interaction regulator, for example procaine hydrochloride (SP-01 and SP-01A); Integrase inhibitor includes but not limited to, L-870 and 810; RNA enzyme H inhibitor; Rev or REV inhibitor; The vif inhibitor (for example come from the proline rich of vif peptide, come from the peptide of HIV-1 protease N end); Virus processing inhibitor includes but not limited to betulinol and dihydro betulin derivatives (for example PA-457); And immunomodulator, include but not limited to AS-101, granulocyte macrophage colony stimulating factor, IL-2, valproic acid and Thymopentin.

In one embodiment, the invention provides compositions, wherein said bioactive molecule (for example antiviral peptide) is dissolved.

In another embodiment, the invention provides compositions, wherein said bioactive molecule (for example antiviral peptide) is suspended.

In another embodiment, the invention provides compositions, the bioactive molecule of wherein said suspension (for example antiviral peptide) is a spray-dried forms.

In another embodiment, the invention provides compositions, the bioactive molecule of wherein said suspension (for example antiviral peptide) is for comprising the spray-dried forms of salt (for example slaine).

In another embodiment, the invention provides compositions, the bioactive molecule of wherein said suspension (for example antiviral peptide) is the zinciferous spray-dried forms of bag.

In another embodiment, the invention provides compositions, the bioactive molecule of wherein said suspension (for example antiviral peptide) is the calcareous spray-dried forms of bag.

In another embodiment, the invention provides compositions, the bioactive molecule of wherein said suspension (for example antiviral peptide) is the ferruginous spray-dried forms of bag.

In another embodiment, the invention provides compositions, the bioactive molecule of wherein said suspension (for example antiviral peptide) is a precipitation form.

In another embodiment, the invention provides compositions, the bioactive molecule of wherein said suspension (for example antiviral peptide) is for comprising the precipitation form of salt (for example slaine).

In another embodiment, the invention provides compositions, the bioactive molecule of wherein said suspension (for example antiviral peptide) is the zinciferous precipitation form of bag.

In another embodiment, the invention provides compositions, the bioactive molecule of wherein said suspension (for example antiviral peptide) is the calcareous precipitation form of bag.

In another embodiment, the invention provides compositions, the bioactive molecule of wherein said suspension (for example antiviral peptide) is the ferruginous precipitation form of bag.

In another embodiment, the invention provides compositions, the bioactive molecule of wherein said suspension (for example antiviral peptide) is the magniferous precipitation form of bag.

In another embodiment, the invention provides compositions, the bioactive molecule of wherein said suspension (for example antiviral peptide) is the copper bearing precipitation form of bag.

In another embodiment, the invention provides compositions, the bioactive molecule of wherein said suspension (for example antiviral peptide) is the aluminiferous precipitation form of bag.

In another embodiment, the invention provides compositions, said composition comprises the salt (for example slaine) that is about 1-15%, 1-14%, 1-13%, 1-12%, 1-11%, 1-10%, 1-9%, 1-8%, 1-7%, 1-6%, 1-5%, 1-4%, 1-3%, 1-2%, 5-15%, 7-15%, 5-10%, 7-10% or 10-15%.In further embodiment, the invention provides the compositions that comprises salt (for example slaine), described salt and biomolecule (for example antiviral peptide) exist with mol ratio at 1: 1.The slaine object lesson that can be used for compositions provided by the invention includes but not limited to, zinc, calcium and ferrum.

By changing the ratio of Binder Materials in the compositions provided by the invention, can regulate the delivery rate of (for example improve or optimize) biomolecule from substrate, described substrate forms after using compositions provided by the invention to the patient.In specific embodiments, reduce the SAIB in the compositions provided by the invention: the PLA ratio can cause biomolecule to raise at any special time in the intravital plasma concentration of patient, and can prolong the time that the specific blood plasma level of biomolecule keeps in patient's body.

In specific embodiments, use NMP to replace glyceryl triacetate or benzyl benzoate can cause biomolecule to raise at any special time, and can prolong the time that the specific blood plasma level of biomolecule keeps in patient's body in the intravital plasma concentration of patient as the solvent in the compositions provided by the invention.In specific embodiments, use NMP to replace glyceryl triacetate can cause C as solvent _MaxRaise.

In another embodiment, increase using volume injected (for example doubling) and can causing biomolecule to raise at any special time of compositions provided by the invention, and can prolong the time that the specific blood plasma level of biomolecule keeps in patient's body in the intravital plasma concentration of patient.

In another embodiment, the type of employed Binder Materials (for example PLA) can influence the pharmacokinetic parameter of compositions provided by the invention.In specific embodiments, the PLA type is changed into 3M from 3L can cause C _MaxReduction, t _MaxIncrease and t _0.01Increase.

In another embodiment, change the delivery rate that the ratio of Binder Materials and solvent in the compositions provided by the invention can influence biomolecule.In specific embodiments, the raising Binder Materials can cause C to the ratio (for example PLGA1A is to NMP) of solvent in compositions provided by the invention _MaxReduction, t _MaxIncrease and t _0.01Increase.

In another embodiment, change the delivery rate that polymer type and molecular weight can improve biomolecule.In specific embodiments, (lactide: Acetic acid, hydroxy-, bimol. cyclic ester) ratio can cause C to increase polymer molecular weight and/or increase L: G _MaxReduction, t _MaxIncrease and t _0.01Increase.

In another embodiment, the peptide concentration (for example doubling) that improves in the compositions provided by the invention can cause C _MaxReduction, t _MaxIncrease and t _0.01Reduce.

In another embodiment, the amount that improves the Binder Materials (for example PLGA) in the described carrier can cause C _MaxReduction, t _MaxIncrease and t _0.01Reduce.

In another embodiment, the amount of increase Binder Materials (for example PLA) can be slowed down the release of biomolecule from compositions provided by the invention and (for example be reduced C in described carrier (carrier that for example comprises SAIB) _Max, increase t _MaxAnd/or increase t _0.01).In specific embodiments, the amount of the PLA in the described carrier is increased to 5% from 1% can further slows down the release of biomolecule from compositions provided by the invention.In another embodiment, the amount of the PLA in the described carrier is increased to 10% from 5% and can further slows down the release of biomolecule from compositions provided by the invention.

Solvent

The solvent that can be used for compositions provided by the invention and method comprises can fully dilute Binder Materials to allow any liquid that can be miscible with water to the described compositions of patient infusion.In one embodiment, described solvent is N-N-methyl-2-2-pyrrolidone N-(NMP).Other suitable solvent includes but not limited to, water, alcohols (for example methanol, ethanol, isopropyl alcohol and benzyl alcohol), glycols (for example Polyethylene Glycol, propylene glycol and tetraethylene glycol (TEG)), benzoate (for example ethyl benzoate and benzyl benzoate), glyceride (for example monoglyceride, diglyceride, triglyceride), glyceryl triacetate and pharmacy acceptable ester (for example ethyl lactate and propyl carbonate).

Binder Materials

The Binder Materials that can be used for compositions provided by the invention and method comprises and can form any material that can be miscible with solvent of substrate by solvent-subcutaneous fluid exchange.In one embodiment, described Binder Materials is Sucrose acetoisobutyrate (SAIB) or derivatives thereof, for example acetic acid sucrose ester or superfine Sucrose acetoisobutyrate (SAIB-SG).In another embodiment, described Binder Materials is polylactide (PLA), for example PLA3L or PLA3M.In another embodiment, described Binder Materials is polylactide-co-glycolide copolymer (PLG, PLGA, PLGA-glucose or derivatives thereof, for example PLGA-PEG1500 or PLA-PEG1500).In another embodiment, described Binder Materials is polycaprolactone or derivatives thereof or its poly (lactide-co-glycolide).The difference of PLA and PLGA is ratio, molecular weight and the end group of lactide/glycolides.Molecular weight is with the digital classification in the title.The molecular weight valuation is 10000 times of described numeral.Described end group is carboxylic acid (A), methyl ester (M) or Laurel alcohol ester (L).

In one embodiment, compositions provided by the invention can comprise two or more different Binder Materials that exist with identical or different percentage by weight.In specific embodiments, described Binder Materials is selected from the mixtures of material of PLA, PLG, PLGA or PLGA-glucose for two or more.

In certain embodiments, the amount of described Binder Materials existence is about 5-95% weight, about 5-90% weight, about 10-90% weight, about 10-85% weight, about 15-85% weight, about 20-85% weight, about 30-85% weight, about 30-80% weight, about 30-70% weight, about 30-65% weight, about 30-60% weight, about 40-85% weight, about 45-85% weight, about 50-85% weight, about 55-85% weight, about 60-85% weight, about 65-85% weight, about 70-85% weight, about 75-85% weight, about 80-85% weight, about 1-15% weight, about 5-15% weight or about 10-15% weight.

In another embodiment, the amount of described Binder Materials existence is about 25-900mg/g, 100-900mg/g, 200-900mg/g, 300-900mg/g, 400-900mg/g, 500-900mg/g, 100-800mg/g, 100-700mg/g, 100-600mg/g, 100-500mg/g, 200-800mg/g, 300-600mg/g, 25-250mg/g, 25-200mg/g, 25-150mg/g, 50-150mg/g, 50-100mg/g, 50mg/g, 75mg/g or 100mg/g.

Peptide

For the antiviral peptide bioactive molecule, any antiviral peptide known in the art all can be used for compositions provided by the invention and method.In one embodiment, described antiviral peptide is the derivant of T20, T1249, T897, T2635, T999 or T1144 or above-mentioned peptide.

The specific antiviral peptide that can be used for compositions provided by the invention and method comprises the HIV fusion inhibitor peptides that is derived from primary amino acid sequence (" basic sequence "), described basic sequence has the aminoacid sequence of SEQ ID NO:5, but the different leucine zipper sample motifs that are to have in its aminoacid sequence more than of every kind of HIV fusion inhibitor peptides and basic sequence wherein, and in its aminoacid sequence, except that forming the necessary leucine of leucine zipper sample motif, has at least one extra leucine (i.e. aminoacid except that the amino acid sites 1 or 8 of leucine zipper sample motif in described sequence; For example replace isoleucine) at the amino acid sites 21 usefulness leucines of SEQ IDNO:5.

Other antiviral peptide that can be used for compositions provided by the invention and method comprises the HIV fusion inhibitor peptides that is derived from primary amino acid sequence (" basic sequence "), described basic sequence has the aminoacid sequence of SEQ ID NO:5, but the different leucine zipper sample motifs that are to have in its aminoacid sequence more than two of every kind of HIV fusion inhibitor peptides and basic sequence wherein.

Other antiviral peptide that can be used for compositions provided by the invention and method comprises a series of HIV fusion inhibitor peptides, wherein every kind of HIV fusion inhibitor peptides: (a) comprise the aminoacid sequence that is derived from HIV gp41 HR2 zone; (b) has the aminoacid sequence that is no less than 2 and no more than 5 leucine zipper sample motifs; (c) in its aminoacid sequence, except that the amino acid sites 1 or 8 of leucine zipper sample motif, have at least one extra leucine (for example, with SEQ ID NO:5-7 in any one or a plurality of sequences basic sequence relatively); And randomly (d) demonstrating beyond thought improvement aspect one or more biological properties.In one embodiment, described HIV fusion inhibitor peptides comprises the aminoacid sequence that is derived from HIV gp41 HR2 zone, and wherein said aminoacid sequence comprises HR2 leucine zipper sample motif, for example the HR2 leucine zipper sample motif of Fig. 1 or Fig. 2 demonstration.In preferred embodiments, described HIV fusion inhibitor peptides length is 14 to 60 amino acid residues.In one embodiment, described HIV fusion inhibitor peptides further comprise N-terminal blocking groups or C-terminal blocking groups or above-mentioned both; Described endcapped group can include, but are not limited to: the amino or the acetyl group that are positioned at N-terminal; And the carboxyl or the amide groups that are positioned at C-terminal.

Other antiviral peptide that can be used for compositions provided by the invention and method comprises the HIV fusion inhibitor peptides, wherein every kind of HIV fusion inhibitor peptides: (a) comprise the aminoacid sequence that is derived from HIV gp41 HR2 zone; (b) has the aminoacid sequence that contains more than 2 and no more than 5 leucine zipper sample motifs; And (c) in its aminoacid sequence, except that the amino acid sites 1 or 8 of leucine zipper sample motif, have at least one extra leucine (for example, with SEQ ID NO:5-7 in any one or a plurality of sequences basic sequence relatively); And (d) demonstrating beyond thought improvement aspect one or more biological properties.In one embodiment, described HIV fusion inhibitor peptides comprises the aminoacid sequence that is derived from HIV gp41 HR2 zone, and wherein said aminoacid sequence comprises HR2 leucine zipper sample motif, for example the HR2 leucine zipper sample motif of Fig. 1 or Fig. 2 demonstration.In preferred embodiments, described HIV fusion inhibitor peptides length is 14 to 60 amino acid residues.In one embodiment, described HIV fusion inhibitor peptides further comprise N-terminal blocking groups or C-terminal blocking groups or above-mentioned both; Described end group can include but not limited to: the amino or the acetyl group that are positioned at N-terminal; And the carboxyl or the amide groups that are positioned at C-terminal.

Other antiviral peptide that can be used for compositions provided by the invention and method comprises the HIV fusion inhibitor peptides that has to the similar aminoacid sequence of SEQ IDNO:5, but described HIV fusion inhibitor peptides aminoacid sequence: (a) have, and (promptly except that 1 or 8 sites of leucine zipper sample motif) have at least one extra leucine except forming the needed leucine of leucine zipper sample motif more than a leucine zipper sample motif; Or (b) have more than 2 leucine zipper sample motifs; And wherein said HIV fusion inhibitor peptides is demonstrating improvement aspect one or more biological properties.In one embodiment, described HIV fusion inhibitor peptides length is 14 to 60 amino acid residues.In one embodiment, described HIV fusion inhibitor peptides comprises the aminoacid sequence that is derived from HIV gp41 HR2 zone, and wherein said aminoacid sequence comprises HR2 leucine zipper sample motif, for example the HR2 leucine zipper sample motif of Fig. 1 or Fig. 2 demonstration.

Other antiviral peptide that can be used for compositions provided by the invention and method comprises for example peptide shown in the SEQ IDNO:9,10,14 and 15, perhaps with SEQ ID NO:9,10,14 and 15 in arbitrary sequence compare the HIV fusion inhibitor peptides that comprises 1 to 3 aminoacid difference.

Other antiviral peptide that can be used for compositions provided by the invention and method comprises the HIV fusion inhibitor peptides of aminoacid sequence and SEQ ID NO:5 primary amino acid sequence similarity, but compare with described primary amino acid sequence, described HIV fusion inhibitor peptides aminoacid sequence has in its aminoacid sequence more than 2 leucine zipper sample motifs; Wherein said HIV fusion inhibitor peptides is demonstrating beyond thought improvement aspect one or more biological properties.

Other antiviral peptide that can be used for compositions provided by the invention and method comprises for example peptide shown in the SEQ IDNO:11-13, perhaps with SEQ ID NO:11-13 in arbitrary sequence compare the HIV fusion inhibitor peptides that comprises 1 to 3 aminoacid difference.

HIV fusion inhibitor peptides of the present invention can be by the conventional preparation of the method known, and described method comprises nucleic acid recombinant expressed of the described peptide of encoding.For example, the engineering cell that is used for recombinant expressed HIV fusion inhibitor peptides can be cultivated reasonable time under suitable condition expressing described peptide, and described peptide can obtain thus.HIV fusion inhibitor peptides of the present invention also can prepare by synthetic method.

It below is the concrete fragments of peptides that can be used for compositions of the present invention.Each fragments of peptides can be as intermediate, thereby this intermediate can produce the HIV fusion inhibitor peptides of the aminoacid sequence with SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15 with one or more other fragments of peptides covalent bond in one group of fragments of peptides.In one embodiment, in one group of fragments of peptides, thereby fragments of peptides combination in a certain way in liquid phase process produces the HIV fusion inhibitor peptides of the required aminoacid sequence with SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.Zhi Bei HIV fusion inhibitor peptides also can be used for compositions provided by the invention in the following way: the component peptide fragment of synthetic described HIV fusion inhibitor peptides, then described fragments of peptides is assembled into described HIV fusion inhibitor peptides, wherein said HIV fusion inhibitor peptides has the aminoacid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.

In one embodiment, the invention provides the method for preparing peptide, described method comprises described peptide spray drying.For example, peptide can wherein use suitable acid or alkali for example 1N NaOH or 1N HCl adjusting pH at pH less than 4 or greater than dissolving (for example in water) in 6 o'clock, then described peptide solution is sprayed onto in the casing of heating by spray nozzle.Can artificially collect exsiccant peptide particles.

In another embodiment, above-mentioned spray drying process further comprises to described spray drying soln and adds excipient, mixes excipient and peptide thus.The example of Exemplary excipients includes but not limited to, filler, extender, diluent, wetting agent, solvent, emulsifying agent, antiseptic, flavoring agent, absorption enhancer, continues release matrix, coloring agent and the macromolecular substances for example albumin or the material of aminoacid and sugar for example.

In another embodiment, the invention provides the method for preparing peptide, described method comprises peptide solution is sprayed onto in (similar to the spray drying mode) another solution (for example metal salt solution, for example zinc salt, iron salt or calcium salt soln) by spray nozzle.Can the gained suspension is centrifugal, abandoning supernatant, freezing precipitation.But described precipitation lyophilizing is also crossed 200 μ m sieve.

In another embodiment, the invention provides the method for preparing peptide, described method comprises salt precipitation or pH precipitation, wherein peptide can be at pH less than 4 or greater than dissolving (for example in water) in 6 o'clock, and for example 1N NaOH or 1N HCl regulate pH between about 4 to 6 or between about 4.8 to 5.2 wherein to use suitable acid or alkali.In specific embodiments, described method comprises in peptide solution and to add saline solution or strong acid/aqueous slkali to cause precipitation.In another embodiment, described method further comprises by centrifugal collecting precipitation, precipitates and randomly will precipitate 200 μ m sieve with the control granular size by lyophilization.

Bound by theory not thinks that the method and the reagent of the peptide that is used to prepare compositions provided by the invention can produce the pharmacokinetic parameter (for example amount of delivery of biologically active molecule or persistent period) that is of value to the ideal of some patient or disease or improves.Especially, metal (for example zinc, ferrum or change) is attached to the pharmacokinetic parameter that mode in the peptide precipitation (for example spray and/or precipitate) can influence resulting composition.

In one embodiment, in described peptide precipitation process, increase described tenor (for example zinc content) and can reduce C _Max, increase t _MaxAnd increase t _0.01In another embodiment, the slaine (for example zinc sulfate) that adds as lyophilizing salt to low metal (for example low zinc) precipitation can reduce C _Max, increase t _MaxAnd increase t _0.01

In another embodiment, the solution that is settled out peptide can influence C _MaxAnd t _MaxFor example, from 50: 50 methanol: be settled out peptide the water with respect to from pure water, being settled out peptide and can reduce C _MaxAnd increase t _Max

In another embodiment, the mode of preparation peptide can influence C _MaxAnd t _{Max is big}For example, the spraying precipitated phase precipitates the C that can raise for non-spraying _MaxAnd increase t _Max

Using method

The present invention further provides and used method for compositions provided by the invention.In one embodiment, described compositions is used as for example part of antiviral therapy scheme of therapeutic scheme.In certain embodiments, such therapeutic scheme is passable, for example, is used to treat HIV and infects.

In one embodiment, the invention provides and use compositions provided by the invention to suppress the method that HIV transmits to target cell, described method comprises to the patient uses a certain amount of compositions provided by the invention, so that described target cell is contacted by the active agents of the effective dose of viral infection (for example antiviral peptide) with the inhibition cell.Described method is passable, for example is used for the treatment of the HIV infected patient.In one embodiment, suppressing HIV comprises HIV-1 and the target cell fusion that suppresses the gp41 mediation and/or suppresses the HIV infection cell and the formation of target cell syncytium to the target cell transmission.

The method that the present invention also provides treatment HIV to infect (in one embodiment, HIV-1 infects), described method comprise to the HIV infected patient uses compositions provided by the invention with the effective dose that treatment HIV infects.In one embodiment, described compositions comprises and suppresses the HIV fusion inhibitor peptides of HIV to the effective dose of target cell transmission, and/or suppresses the HIV of gp41 mediation and the HIV fusion inhibitor peptides of the effective dose that target cell is merged.Described method is passable, for example, is used for the treatment of the HIV infected patient.

In specific embodiments, the invention provides and improve HIV and infect related indication method, described method comprises to the HIV infected patient uses the compositions that comprises solvent, Binder Materials and peptide, wherein said peptide is selected from T20, T1249, T897, T2635, T999 and T1144, or the combination of above-mentioned peptide.

The present invention further provides the method that the compositions that will comprise the HIV fusion inhibitor peptides is used to prepare the medicine (for example being used to suppress method, the method for inhibition HIV fusion and/or the method that treatment HIV infects that HIV transmits) that is used for the treatment of HIV and infects.Described medicine can be pharmaceutical compositions, and described pharmaceutical composition comprises bioactive molecule for example HIV fusion inhibitor peptides, solvent, Binder Materials, and randomly comprises one or more pharmaceutical acceptable carriers.

In one embodiment, compositions provided by the invention can be used by injection, for example subcutaneous injection.

In another embodiment, compositions provided by the invention can be used (for example passing through subcutaneous injection) once in per 3,5,7,10,14,17,21,28 or 60 days.

In another embodiment, sustainable one day of compositions provided by the invention or many days, a week or many weeks, January or many months or 1 year or use (for example passing through subcutaneous injection) for many years every day 1 time, 2 times, 3 times or more times.

In another embodiment, compositions provided by the invention can be used (for example passing through subcutaneous injection) weekly 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times or more times.In specific embodiments, compositions provided by the invention can be used (for example passing through subcutaneous injection) weekly 1 time or 2 times.In another particular, per 2 weeks of compositions provided by the invention are used (for example passing through subcutaneous injection) 2 times.Use at every turn and can comprise 1 time, 2 times, 3 times or more times injection.

In one embodiment, the applied volume of compositions provided by the invention is 100 μ l, 200 μ l, 300 μ l, 400 μ l, 500 μ l, 600 μ l, 700 μ l, 800 μ l, 900 μ l, 1000 μ l or more.

Embodiment

Embodiment 1

In following examples, multiple biophysics mathematic(al) parameter and biological parameter have been assessed.The method of determining described parameter is as described below.

Use the standard solid-phase synthetic technology and use standard FMOC chemistry of peptides method, or synthesize peptide on the peptide synthesizer, comprise HIV fusion inhibitor peptides and basic sequence as solid phase synthesis and synthetic being combined in of liquid phase that the embodiment of the invention 3 describes in detail.In the present embodiment, described HIV fusion inhibitor peptides can further comprise reactive functional groups, and promptly most of described reactive functional groups is protected by acetyl group and/or protected by amide groups at the C end at the N end.After the resin disengaging, described peptide is precipitated, and described precipitation is by lyophilizing.Use the described peptide of reversed-phase high-performance liquid chromatography purification then; Confirm the identity of peptide with electron spray mass spectrometry.

The assessment of biophysical parameters comprises measures helicity and heat stability.Use the following mensuration helicity of circular dichroism spectra (" CD ").Briefly, use the spectrometer of being furnished with the thermoelectric control device to obtain CD spectrum.Spectrum acquisition condition is: 25 ℃, and from 200 to 260nm steppings, 0.5 nanometers (nm), bandwidth 1.5nm, 4 seconds typical mean time/step.After deduction cell/buffer blank, use conservative window size by the level and smooth spectrum of three rank least square polynomial fits to provide random residual.Original ellipticity numerical value uses standard method to be converted to mean residue ellipticity, and with wavelength (200 arrive 260nm) to [θ] * 10 ^-3(degree cm ²/ dmol) mapping.Use standard method to calculate percentage ratio helicity value (being typically expressed as 10 μ M, 25 ℃ percentage ratio helicity) then.Thermal stability evaluation is following to carry out: monitoring CD signal is in the variation at 222nm place when with 2 ℃ of stepping elevated temperatures, and equilibration time is 1 minute.The heat stability (with the Tm value representation) of each sample (for example HIV fusion inhibitor peptides) is corresponding to the peaked temperature of heat deflection first derivative.

The assessment of biological property comprises the antiviral activity of mensuration to the HIV-1 strain.At definite HIV fusion inhibitor peptides antiviral activity (for example, a kind of measurement is to suppress the ability that HIV transmits to target cell) time, used analyzed in vitro, come from the data that the peptide in HIV gp41HR zone produces by use, described analyzed in vitro has shown that observed antiviral activity in the body is had predictability.More specifically, for the identical peptide that comes from HIV gp41, use Infection in Vitro analysis (" Magi-CCR5 analysis of infection ", referring to for example U.S. Patent number 6,258,782) observed antiviral activity has demonstrated active rationally relevant (referring to, people such as Kilby for example with interior resisting virus, 1998, Nature Med.4:1302-1307).Described analysis uses the derive CCR5 of cMAGI of indicating clone MAGI or expression the infectious virus titre is reduced to keep the score.Beta galactosidase (the β-gal) the expression of reporter gene that above-mentioned two kinds of cell lines all utilize the ability of HIV-1 tat to drive with trans-activation HIV-LTR.Described β-gal reporter gene has been modified being positioned in the nucleus, and can detect with the strong nuclear staining of X-gal substrate in several days after infection.If have only the infection of wheel before the dyeing, the nuclear volume that is colored just may be interpreted as the infectious virus number of particles that equals in the provocative inoculation thing (challengeinoculum).The cell of infection all demonstrates the linear relationship between the infection cell quantity that virus is imported and imager shows with CCD-imager counting, the separated strain of primary cell and laboratory change.In MAGI and cMAGI analysis, it is significant that infection titer reduces by 50% (Vn/Vo=0.5), and the main cutoff value (" IC50 " is defined as and causes the infectious virus titre to reduce by 50% activity component concentration) of assessment antiviral activity is provided.The peptide that is used to test antiviral activity is diluted into multiple concentration, and inoculation is carried out 2 times or 3 retests at HIV, and described HIV inoculum produces about 1500 to 2000 infection cell/holes in being adjusted at 48 hole microtitration plates.Described peptide (in corresponding dilution) is added in cMAGI or the MAGI cell, is the virus inoculation thing then; After 24 hours, add infection and cell-cell fusion inhibitor (for example SEQ ID NO:2 (En Fuwei ground)) to prevent that second takes turns HIV infection and cell-cell virus propagation.Described cell was cultivated 2 days again, fixing then and with the X-gal substrate staining with detection HIV infection cell.The infection cell quantity of each contrast and peptide dilution is determined with the CCD imager, calculates IC50 (representing with μ g/ml) then.

The virus that the antiviral activity of the peptide be made up of basic sequence is had a resistance can be used the laboratory method preparation of standard.Basic, after calculating IC50 and IC90, cell mixes (comprising when after this described cell divides bottle) in culture with viral and peptide (for example concentration is near IC90).Keeping and monitor described culture occurs until syncytium.The virus of cultivating results from the first round is used for taking turns the cultivation infection cell second, and the concentration of peptide is higher than the concentration that the cultivation of (2 to the 4 times) first round is used.Keep and monitor second and take turns the existence that in the culture antiviral activity of described peptide is had the virus of resistance.May cultivate the virus isolated strain (at the IC50 predeterminated level of the peptide that resists this type of separated strain) that the antiviral activity of described peptide is had resistance with final generation with the later several rounds.

For determining pharmacokinetic property, HIV fusion inhibitor peptides or the basic sequence intravenous that derives the HIV fusion inhibitor peptides are administered to macaque (Macaca fasicularis) (as known in the art, other animal model also can be used for determining pharmacokinetic property).A plurality of time points are taken a blood sample and centrifugal separation plasma after using.Freezing preservation plasma sample is analyzed until carry out LC-MS (liquid chromatography/mass spectrometry) under the electron spray cation mode.Use acetonitrile gradient in the 10mM ammonium acetate buffer of pH6.8 with HIV fusion inhibitor or basic sequence eluting on C18 or the C8HPLC post.During analysis, the acetonitrile that comprises 0.5% formic acid that uses 2 times or 3 times volumes is with the plasma sample Deproteinization.The double calibration criterion and the described sample of macaque plasma sample prepare simultaneously, and analyze before or after the sample of the described HIV of comprising fusion inhibitor peptides or basic sequence.Use single index or two index mathematical model to calculate pharmacokinetic property from plasma concentration-time data.Obtain model by non-linear least square optimization.Used concentration 1/C ²Weight.Use area (AUC) under following Equation for Calculating plasma concentration-time graph, systemic clearance (Cl) and the terminal half-life (t of elimination ¹/ ₂).

AUC＝A/-a+B/-b

Wherein A and B are intercept, and a and b are the speed constant of exponential equation, describe distribution phase and elimination phase respectively.When using Single-Index Model, eliminate parameter " A " and " a ".

Cl=dosage/AUC (representing) with L/K/hr

t ¹/ ₂=-0.6903/b (with hour (hr) expression)

Embodiment 2

For embodiment provided by the invention is described, basic sequence has following aminoacid sequence (SEQ IDNO:5).

TTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALREL

In one embodiment, the HIV fusion inhibitor peptides is compared with the basic sequence in its source, comprises more than 2 leucine zipper sample motifs.The example of these HIV fusion inhibitor peptides includes but not limited to, SEQ IDNO:11, SEQ ID NO:12 and SEQ ID NO:13; Or with SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13 in arbitrary sequence compare aminoacid sequence with 1 to 3 aminoacid difference (for example, at least 92% concordance); Each HIV fusion inhibitor peptides has the aminoacid sequence of 3 to 5 leucine zipper sample motifs.Below diagram (I) has shown the aminoacid sequence of HIV fusion inhibitor peptides, (with " | " alignment) encodes with single-letter aminoacid below the amino acid sites of described basic sequence, and (" L " represents leucine, " I " represents isoleucine) expression aminoacid difference (comparing) with basic sequence, and the isoleucine of participation leucine zipper sample motif and leucine (in 1 or 8 sites of same leucine zipper sample motif, or 1 site of 8 sites of a leucine zipper sample motif and adjacent leucine zipper sample motif) indicate with underscore.1 or 8 sites of a leucine zipper also can be used as the relative end site of another leucine zipper sample motif in the sequence, promptly as 8 sites of a motif and 1 site of another adjacent motif.

In another embodiment, the HIV fusion inhibitor peptides is compared with the basic sequence in its source, comprise more than 1 leucine zipper sample motif, and the extra leucine that does not participate in forming leucine zipper sample motif.The example of these HIV fusion inhibitor peptides includes but not limited to, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:14 and SEQ ID NO:15; Or with SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:14 or SEQ ID NO:15 in arbitrary sequence compare aminoacid sequence with 1 to 3 aminoacid difference (for example, at least 92% concordance); And the difference of every kind of HIV fusion inhibitor peptides aminoacid sequence and SEQ ID NO:5 basic sequence is to comprise more than 1 leucine zipper sample motif, and the extra leucine (that is the leucine except that leucine zipper sample motif 1 or 8 sites) that does not participate in forming leucine zipper sample motif.In one embodiment, non-leucine zipper sample motif leucine is replaced and has been replaced isoleucine at the amino acid sites 21 of SEQ ID NO:5 basic sequence, and this replacement provides the factor of the favourable biological property that promotes described peptide.Below diagram (II) has shown the aminoacid sequence of HIV fusion inhibitor peptides, (with " | " alignment) encodes with single-letter aminoacid below the amino acid sites of described basic sequence, and (" L " represents leucine, " I " represents isoleucine) expression aminoacid difference (comparing) with basic sequence, and the isoleucine of participation leucine zipper sample motif and leucine (in 1 or 8 sites of same leucine zipper sample motif, or 1 site of 8 sites of a leucine zipper sample motif and adjacent leucine zipper sample motif) indicate with underscore.The leucine that does not participate in forming leucine zipper sample motif is an italic.

Below diagram (III) has shown the relatively summary of " basic sequence " SEQ ID NO:5 and SEQ IDNO:9-15 peptide disclosed by the invention, and SEQ ID NO:9-15 peptide demonstrates the biological property of improvement with respect to SEQ ID NO:5.Each sequence is replaced with underscore and runic with respect to the aminoacid of SEQ ID NO:5 basic sequence and is indicated among the SEQ ID NO:9-15.

Reference table 1 will compare according to HIV fusion inhibitor peptides of the present invention and synthetic peptide, and described synthetic peptide has identical basic sequence, but aminoacid sequence different (comparing with SEQ ID NO:9-15), and have HIV (human immunodeficiency virus)-resistant activity.Described biophysical parameters and the biological activity parameter of determining with the embodiment of the invention 1 described method that relatively comprise.When determining biological activity, owing to assess by antiviral activity, used virus isolated strain, described virus isolated strain has resistance (described resistance virus isolated strain is called after " Res " in table 1) to the antiviral activity of some peptide of the fusion of known inhibition HIV mediation.

Table 1: biophysics and biology (antiviral activity) parameter

??SEQ?ID??NO：	Helicity (%)	??Tm(℃)	Antiviral activity (μ g/ml) HIV-IIIB IC50	Antiviral activity (μ g/ml) HIV-Res IC50
					??5	??71	??42	??＜0.10	??＜0.10
??6	??97	??65	?＜?0.10	??≥0.10
					??7	??84	??75	??＜0.10	??＞0.10
??8	??99	??46	??＜0.10	Not test
					??9	??61	??62	??＜0.10	??＜0.10
??10	??77	??75	??＜0.10	??＜0.10

SEQ ID NO:6 and SEQ ID NO:7 and basic sequence SEQ ID NO:5 different are that single leucine replaces (laying respectively at 24 sites and 31 sites); Shown in above table 1, described replacement does not influence antiviral activity significantly, but has caused improve (referring to the following table 2) of half-life.SEQ ID NO:6 is similar to the HIV fusion inhibitor peptides that has SEQ ID NO:9 according to the present invention, but the aminoacid sequence of SEQ ID NO:9 has another aminoacid difference, the leucine of amino acid sites 21 (and SEQ IDNO:6 is isoleucine at amino acid sites 21).Reference table 1, with the peptide comparison of SEQ ID NO:6, leucine replacement isoleucine causes helicity to reduce (from 97% to 61%) among the SEQ ID NO:9, keeps good resistance character (activity of antagonism virus isolated strain " Res ") simultaneously.Similarly, the aminoacid sequence of SEQ ID NO:7 is similar to the HIV fusion inhibitor peptides that has SEQ ID NO:10 according to the present invention, but the aminoacid sequence of SEQ ID NO:10 has an aminoacid difference, the leucine of amino acid sites 21 (and SEQ ID NO:7 is isoleucine at amino acid sites 21).Reference table 1, with the peptide comparison of SEQ ID NO:7, leucine replacement isoleucine causes helicity to reduce (from 84% to 77%) among the SEQ ID NO:10, keeps good resistance character (activity of antagonism virus isolated strain " Res ") simultaneously.Therefore, table 1 has shown SEQ ID NO:9 and 10 character of improving with respect to SEQ ID NO:6-7.

The present embodiment has illustrated with the primary amino acid sequence and has compared, according to the pharmacokinetic property of HIV fusion inhibitor peptides of the present invention.The method of the assessment pharmacokinetic property that use embodiment 1 describes in detail before this, table 2 has illustrated with the pharmacokinetic property of basic sequence SEQ ID NO:5 and has compared, according to the pharmacokinetic property of HIV fusion inhibitor peptides of the present invention.

Table 2

??SEQ?ID?NO：	Clearance rate (L/kg/hr)	Half-life (t 1/ 2：hr)
			??5	??＞0.04	??6
??6	??＜0.02	??15
			??7	??＜0.02	??17
??8	??＞0.04	??7
			??9	??＜0.02	??12
??10	??＜0.02	??21

As shown in table 2, SEQ ID NO:6,7,9 and 10 demonstrates the biological half-life (" t of prolongation ¹/ ₂").SEQ ID NO:8 is at amino acid sites 21 but not amino acid sites 24 or 31 is a leucine, do not demonstrate the half-life of the shown significant prolongation of SEQ ID NO:6,7,9 and 10 peptides.

For the HIV fusion inhibitor peptides being formulated in the pharmaceutical acceptable carrier when the production pharmaceutical preparation, the stability in aqueous solution may be important parameters, especially when pharmaceutical preparation during by parenteral administration.It should be noted that HIV fusion inhibitor peptides according to the present invention demonstrates the stability of improving under physiological pH in aqueous solution.For example, following test respectively has the synthetic peptide of SEQ ID NO:2, SEQ ID NO:5 aminoacid sequence, dissolubility with the HIV fusion inhibitor peptides with SEQ ID NO:9 aminoacid sequence: adding concentration in phosphate buffer (PBS) is the described peptide of 10mg/ml, different time points in (168 hours) time in 1 week is measured (for example using HPLC) 37 ℃ of amounts of residual peptide in the solution of about pH 7.3 in about pH 7.5 scopes down.The solution that comprises SEQ ID NO:2 after a few hours only, become unstable (detecting minimum peptide in the solution).On the contrary, the time point in 1 week still can in solution, detect 90% or more HIV fusion inhibitor peptides have SEQ ID NO:9 aminoacid sequence, be less than 80% peptide and have SEQ ID NO:5 aminoacid sequence but in solution, detect only at the time point in 1 week.

Embodiment 3

Biological property and other effective antiviral agent (comprising SEQ ID NO:2 (En Fuwei ground)) of generally acknowledging of HIV fusion inhibitor peptides provided by the invention are compared.Especially, interested noval chemical compound SEQ ID NO:9 and the antiviral agent SEQ ID NO:2 that generally acknowledges have been carried out external resistance comparative study, details are as follows: the MT2 cell infects with virus isolated strain (IIIB, 030,060 and 098), and cultivates with the selection resistance in SEQ ID NO:2 (En Fuwei ground) that concentration raises gradually or SEQ ID NO:9.Initial peptide concentration is about the IC of each peptide to corresponding wild type separated strain ₅₀2 times.Add fresh peptide by every 1-3 days and keep peptide concentration.Use the cytopathic effect (CPE) of standard technique monitoring culture, when reaching maximum CPE, a little equal portions virus is used for the infection of round subsequently.Compare with the wild-type virus growth rate, according to incubation time, peptide concentration raises 2 to 4 times.During selecting, also collect the viral liquid storage that does not contain peptide.The viral liquid storage that does not contain peptide identifies that with the dideoxy sequencing chemical method genotype of gp41 changes, and uses the cMAGI analysis of infection to determine the phenotype susceptibility.

It is as shown in table 3 to use SEQ ID NO:2 and SEQ ID NO:9 to carry out the comparative result of external selection.The incubation time that these data show SEQ ID NO:9 selects is selected average long 3 times than SEQ ID NO:2, causes IC ₅₀Lower multiple changes (42 times of SEQ ID NO:2 with SEQ ID NO:9 24 times compare).The sudden change (geometric mean 3.6) that SEQ ID NO:9 need to select is more manyed to reach described lower multiple change than SEQ ID NO:2 (geometric mean 1.7).Longer incubation time, lower multiple change and realize that described lower multiple changes required higher sudden change quantity, shows all with SEQ ID NO:2 and compare that SEQ ID NO:9 demonstrates the external resistance of more difficult development.That is to say that described presentation of results HIV occurs the resistance of SEQ ID NO:9 is needed the longer time than the resistance that occurs SEQ ID NO:2.To for example HIV resistance development research of SEQ ID NO:2 and T1249 of other peptide, people can expect that in vitro results provided by the invention can be reasonably related with result in the body based in the past.

The comparison of table 3:SEQ ID NO:2 (En Fuwei ground) and the external selection of SEQ ID NO:9

							Initial virus isolated strain	Peptide (SEQ ID NO)	Cultivate natural law	Initial IC50 (ng/mL)	Stop IC50 (ng/mL)	The IC50 multiple changes	Obtain the number of sudden change
??IIIB	??2	??62	??6	??163	??27	??2
							??584.000030	??2	??46	??42	??798	??19	??2
??584.000060	??2	??46	??10	??68	??7	??2
							??584.000098	??2	??45	??50	??45575	??912	??1
Geometrical mean	??2	??49	??19	??797	??42	??1.7
							Initial virus isolated strain	Peptide (SEQ ID NO)	Cultivate natural law	Initial IC50 (ng/mL)	Stop IC50 (ng/mL)	The IC50 multiple changes	Obtain the number of sudden change
??IIIB	??9	??168	??12	??2768	??231	??4
							??584.000030	??9	??77	??28	??113	??4	??2
??584.000060	??9	??173	??8	??208	??26	??4
							??584.000098	??9	??173	??37	??521	??14	??5
Geometrical mean	??9	??140	??18	??429	??24	??3.6

Based in the past to for example HIV resistance development research of SEQ ID NO:2 and T1249 of other peptide, people can expect in vitro results provided by the invention should rationally be associated with result in the body (referring to, people such as Melby for example, 2006, AIDS Research and Human Retroviruses 22 (5): 375-385; Greenberg﹠amp; Cammack, 2004, J.Antimicrobial Chemotherapy 54:333-340; People such as Sista, 2004, AIDS 18:1787-1794).

Embodiment 4

Generally speaking, HIV fusion inhibitor peptides provided by the invention can be by a kind of the synthesizing in two kinds of methods.First method is synthetic for the linearity of using standard solid-phase synthetic technology and use standard Fmoc chemistry of peptides method or other standard peptide chemical method (using CPG).The second method of synthetic HIV fusion inhibitor peptides provided by the invention is for passing through fragment condensation.Briefly, the individual or a plurality of fragments of Synthetic 2, each fragment comprises the appropriate section of the HIV fusion inhibitor peptides complete amino acid sequence that will prepare.In fragment was synthetic, if desired, the aminoacid of adding can have by the chemoprotectant unhindered amina of chemical protective agent (for example pendant amine).Assembling (in a certain way with the order covalent bond) described fragment prepares (with correct aminoacid sequence) described HIV fusion inhibitor peptides thus then.

Synthetic about peptide, the technology that can use the synthetic those skilled in the art of peptide sequence to know prepares each fragments of peptides self, and by the HIV fusion inhibitor peptides provided by the invention of one group of fragments of peptides combinations produce.For example, in one approach, fragments of peptides can synthesize in solid phase, makes up the fusion inhibitor peptides with preparation product HIV then in assembling process in liquid phase.In another approach, can use the synthetic preparation of liquid phase fragments of peptides, described then fragments of peptides makes up the fusion inhibitor peptides with preparation HIV in solid phase in assembling process.In another approach, various fragments of peptides can use solid phase synthesis, make up the complete amino acid sequence with preparation HIV fusion inhibitor peptides then in assembling process in solid phase.In one embodiment, use solid-phase synthesis well known to those skilled in the art to prepare each fragments of peptides.In another embodiment, use one group of fragments of peptides and use solid phase and the preparation of the assembling process of liquid technology combination has the HIV fusion inhibitor peptides of SEQ ID NO:9 aminoacid sequence.For example, one group of fragments of peptides comprises 2 to 4 fragments of peptides, and described fragment is synthesized, and assembling then is to finish the synthetic of HIV fusion inhibitor peptides provided by the invention.Based on instruction of the present invention, it will be apparent for a person skilled in the art that described fragment assemble method can be used for and be used to have some HIV fusion inhibitor peptides of the aminoacid sequence of arbitrary sequence among the SEQ ID NO:9-16.

For explanation prepares HIV fusion inhibitor peptides provided by the invention by fragment condensation, in the method for synthetic HIV fusion inhibitor peptides with SEQ ID NO:9 aminoacid sequence, the fragments of peptides covalent bond in the fragments of peptides assembling process in one group of fragments of peptides.Fragments of peptides provided by the invention can include but not limited to have the fragments of peptides of aminoacid sequence as shown in table 4 below.Some fragments of peptides provided by the invention can be used for getting rid of other fragments of peptides.Also indicated the corresponding aminoacid of various fragments of peptides in SEQ ID NO:9; Therefore, demonstrating various fragments of peptides is made up of a plurality of successive aminoacid in the SEQ ID NO:9 aminoacid sequence.

Table 4

?SEQ?ID?NO：	Aminoacid sequence	Amino acid sites in SEQ ID NO:9
			??17	??TTWEAWDRAIAE	??1-12
??18	??YAARIEALLRALQE	??13-26
			??19	??QQEKNEAALRE	??27-37
??20	??QQEKNEAALREL	??27-38
			??21	??TTWEAWDRAIA	??1-11
??22	??EYAARIEALLRALQE	??12-26
			??23	??TTWEAWDRAI	??1-10
??24	??AEYAARIEALLRALQE	??11-26
			??25	??TTWEAWDRA	??1-9
??26	??IAEYAARIEALLRALQE	??10-26
			??27	??TTWEAWDR	??1-8
??28	??AIAEYAARIEALLRALQE	??9-26
			??29	??TTWEAWDRAIAEYAARIEAL	??1-20
??30	??LRALQEQQEKNEAALRE	??21-37
			??31	??LRALQEQQEKNEAALREL	??21-38
??32	??TTWEAWDRAIAEYAARIE	??1-18
			??33	??ALLRALQEQQEKNEAALRE	??19-37
??34	??ALLRALQEQQEKNEAALREL	??19-38
			??35	??YAARIE?ALLRALQEQQEKNAEAALREL	??13-38
??36	??EYAARIE?ALLRALQEQQEKNEAALREL	??12-38
			??37	??AEYAARIE?ALLRALQEQQEKNEAALREL	??11-38
??38	??IAEYAARIE?ALRALQEQQEKNEAALREL	??10-38
			??39	??AIAEYAARIE?ALLRALQEQQEKNEAALREL	??9-38
??40	??TTWEAWDRAIAEYAARIEALLRALQE	??1-26

The present invention also provides specific fragments of peptides group, described fragments of peptides group in the method for synthetic HIV fusion inhibitor peptides with SEQ ID NO:9 aminoacid sequence as intermediate.Fragments of peptides group provided by the invention comprises 1-16 group, (numbering of group only for convenience of description) as shown in table 5.Some fragments of peptides group can be used for getting rid of other fragments of peptides group.

Table 5

Group number	Fragments of peptides	Amino acid sites in SEQ ID NO:9
			??1	??TTWEWDRAIAE(SEQ?ID?NO：17)??YAARIEALLRALQE(SEQ?ID?NO：18)??QQEKNEAALRE(SEQ?ID?NO：19)	??1-12??13-26??27-37
??2	??TTWEAWDRAIAE(SEQ?ID?NO：17)??YAARIEALLRALQE(SEQ?ID?NO：18)??QQEKNEAALREL(SEQ?ID?NO：20)	??1-12??13-26??27-38
			??3	??TTWEAWDRAIAEYAARIEAL(SEQ?ID?NO：29)??LRALQEQQEKNEAALRE(SEQ?ID?NO：30)	??1-20??21-37
??4	??TTWEAWDRAIAEYAARIEAL(SEQ?ID?NO：29)??LRALQEQQEKNEAALREL(SEQ?ID?NO：31)	??1-20??21-38
			??5	??TTWEAWDRAIA(SEQ?ID?NO：21)??EYAARIEALLRALQE(SEQ?ID?NO：22)??QQEKNEAALRE(SEQ?ID?NO：19)	??1-11??12-26??27-37
??6	??TTWEAWRAI(SEQ?ID?NO：23)??AEYAARIEALLRALQE(SEQ?ID?NO：24)??QQEKNEAALRE(SEQ?ID?NO：19)	??1-10??11-26??27-37
			??7	??TTWEAWDRA(SEQ?ID?NO：25)??IAEYAARIEALLRALQE(SEQ?ID?NO：26)??QQEKNEAALRE(SEQ?ID?NO：19)	??1-9??10-26??27-37
??8	??TTWEAWDR(SEQ?ID?NO：27)??AIAEYAARIEALLRALQE(SEQ?ID?NO：28)??QQEKNEAALRE(SEQ?ID?NO：19)	??1-8??9-26??27-37
			??9	??TTWEAWDRAIA(SEQ?ID?NO：21)??EYAARIEALLRALQE(SEQ?ID?NO：22)??QQEKNEAALREL(SEQ?ID?NO：20)	??1-11??12-26??27-38
??10	??TTWEAWDRAI(SEQ?ID?NO：23)??AEYAARIEALLRALQE(SEQ?ID?NO：24)??QQEKNEAALREL(SEQ?ID?NO：20)	??1-10??11-26??27-38
			??11	??TTWEAWDRA(SEQ?ID?NO：25)??IAEYAARIEALLRALQE(SEQ?ID?NO：28)??QQEKNEAALREL(SEQ?ID?NO：20)	??1-9??10-26??27-38
??12	??TTWEAWDR(SEQ?ID?NO：27)??AIAEYAARIEALLRALQE(SEQ?ID?NO：28)??QQEKNEAALREL(SEQ?ID?NO：20)	??1-8??9-26??27-38
			??13	??TTWEAWDRAIAEYAARIE(SEQ?ID?NO：32)??ALLRALQEQQEKNEAALRE(SEQ?ID?NO：33)	??1-18??19-37
??14	??TTWEAWDRAIAEYAARIE(SEQ?ID?NO：32)??ALLRALQEQQEKNEAALREL(SEQ?ID?NO：34)	??1-18??19-38
			??15	??TTWEAWDRAIAEYAARIEALLRALQE(SEQ?ID?NO：??40)??QQEKNEAALRE(SEQ?ID?NO：19)	??1-26??27-37
??16	??TTWEAWDRAIAEYAARIEALLRALQE(SEQ?ID?NO：??40)??QQEKNEAALREL(SEQ?ID?NO：20)	??1-26??27-38

Therefore, in one embodiment, the invention provides the method, fragments of peptides and the fragments of peptides group that can be used for synthesizing HIV fusion inhibitor peptides with SEQ ID NO:9 aminoacid sequence.It is evident that also that from the present invention describes described method, fragments of peptides and fragments of peptides group can be used for synthetic HIV fusion inhibitor peptides with SEQ IDNO:9 aminoacid sequence, wherein said HIV fusion inhibitor peptides comprises one or more chemical groups:

Wherein one or more amino terminals, carboxyl terminal or side chain dissociate reactive functional groups (for example ε amine of internal lysine) with chemical group (B, U, Z; Wherein B, U, Z can be identical chemical group or different chemical groups) to modify, described chemical group can include but not limited to, one or more following groups: reactive functional groups, chemoproection group (CPG) and joint.The technology that this area is also known to be used in fragments of peptides N end or fragments of peptides C end, to introduce chemical group in unhindered amina or its combination place of internal amino acid.The HIV fusion inhibitor peptides that has SEQ ID NO:9 aminoacid sequence with preparation is relevant, and the illustrative examples of protected fragments of peptides (fragments of peptides with one or more chemical groups) includes but not limited to, listed fragments of peptides in the table 6.

Table 6

?SEQ?ID?NO：	Aminoacid sequence	Amino acid sites in SEQ ID NO:9
			??17	??Ac-TTWEAWDRAIAE	??1-12
??18	??CPG-YAARIEALLRALQE	??13-26
			??19	??CPG-QQEKNEAALRE	??27-37
??19	??CPG-QQEKNEAALRE??I??U	??27-37
			??20	??QQEKNEAALREL-NH 2	??27-38
??29	??Ac-TTWEAWDRAIAEYAARIEAL	??1-20
			??30	??CPG-LRALQEQQEKNEAALRE	??21-37
??31	??LRALQEQQEKNEAALRE?L-NH 2	??21-38
			??21	??Ac-TTWEAWDRAIA	??1-11
??22	??CPG-EYAARIEALLRALQE	??12-26
			??23	??Ac-TTWEAWDRAI	??1-10
??24	??CPG-AEYAARIEALLRALQE	??11-26
			??25	??Ac-TTWEAWDRA	??1-9
??26	??CPG-IAEYAARIEALLRALQE	??10-26
			??27	??Ac-TTWEAWDR	??1-8
??28	??CPG-AIAEYAARIEALLRALQE	??9-26
			??32	??Ac-TTWEAWDRAIAEYAARIE	??1-18
??33	??CPG-ALLRALQEQQEKNEAALRE	??19-37
			??34	??ALLRALQEQQEKNEAALREL-NH 2	??19-38

The Ac-acetyl group, NH ₂-amino (but can be other chemical group, describe in detail as the present invention's " definition " part); CPG is chemoproection group (for example, Fmoc or other N end chemoproection group describe in detail as this paper " definition " part); U as above defines.

Embodiment 5

Reference table 5 (group 1 and group 2) and Fig. 3 have illustrated and have used 3 kinds of particular peptide fragments (SEQ IDNO:17-19+Leu for example; Or SEQ ID NO:17,18 and 20) and use the synthetic method of fragment condensation, described fragment condensation comprises 3 fragments of peptides combinations with preparation HIV fusion inhibitor peptides with HIV fusion inhibitor peptides of SEQ ID NO:9 aminoacid sequence.Physical property that each described fragments of peptides demonstrates and dissolubility property make described fragments of peptides become preferred peptide fragment (with respect to two fragment methods), with the method that is used for synthesizing HIV fusion inhibitor peptides, and further only need a kind of application of sample resin as parent material (simplification synthetic method) with SEQ ID NO:9 aminoacid sequence with high yield and high-purity.12 amino acids are (referring to Fig. 3 before having SEQ ID NO:17 aminoacid sequence and comprise SEQ ID NO:9 by the standard solid-phase synthetic method being synthetic; " AA (1-12) ") fragments of peptides (use the super acid sensitive resin; for example 4-methylol-3-methoxy phenoxy butyrate resin or 2-chlorine trityl chloride resin-" CTC "; Fig. 3); the acetylation (" Ac " of described fragment N end; as chemical group), the C end has hydroxyl (OH) (referring to Fig. 3, " Ac-AA (1-12)-OH ").Have SEQ ID NO:18 aminoacid sequence and comprise among the SEQ ID NO:9 13-26 amino acids by the standard solid-phase synthetic method is synthetic (referring to Fig. 3; " AA (13-26) ") fragments of peptides; described fragment N end has Fmoc (as the chemoproection group); the C end has-OH (referring to Fig. 3, " Fmoc-AA (13-26)-OH ").Have SEQ ID NO:19 aminoacid sequence and comprise among the SEQ ID NO:9 27-37 amino acids by the standard solid-phase synthetic method is synthetic (referring to Fig. 3; " Fmoc-AA (27-37)-OH ") fragments of peptides; described fragment N end has Fmoc (as the chemoproection group); the C end has-OH (referring to Fig. 3, " Fmoc-AA (27-37)-OH ").Use decomposition agent well known to those skilled in the art, solvent and technology with the cracking from the solid phase synthesis resin of each fragments of peptides.Following then each fragments of peptides of separation: remove the major part of above-mentioned solvent by distillation, and precipitate described fragments of peptides by adding water (adding or do not add the cosolvent that contains alcohol).The separation of gained solid by filtration, washing, slurrying again in water or ethanol/water, refilter and dry in vacuum drying oven.

As shown in Figure 3, by the synthetic preparation of liquid phase fragments of peptides, the fragments of peptides that wherein has SEQ ID NO:19 aminoacid sequence is (referring to Fig. 3, " FmocAA (27-37)-OH ") with SEQ ID NO:9 the 38th amino acids leucine (in liquid phase by amidatioon) chemical bond, thereby generate fragments of peptides (comprising 27-38 amino acids among the SEQ ID NO:9) with SEQ ID NO:20 aminoacid sequence, the amidatioon (as chemical group) of described fragments of peptides C end (referring to Fig. 3, " Fmoc-AA (27-38)-NH ₂").In a kind of synthetic method, amidated peptide fragment provided by the invention (includes but not limited to fragments of peptides H-AA (27-38)-NH ₂) can use amide resin directly synthetic.Sum up described liquid phase reactor, the c-terminus of isolating fragments of peptides Fmoc-AA (27-37)-OH is converted into active ester HOBT (I-hydroxybenzotriazole hydrate), 6-Cl HOBT, or HOAT (1-hydroxyl-7-azo benzotriazole), at DIEA (diisopropylethylamine) and leucyl amine (for example, the combination of coupling agent and racemization inhibitor) exists down, HBTU (O-BTA 1-base-N is used in described conversion respectively, N, N ', N '-tetramethylurea hexafluorophosphoric acid ester) or TBTU (O-BTA-1-base-N, N, N ', N '-tetramethylurea Tetrafluoroboric acid ester), and HOBT, 6-Cl HOBT, or HOAT.Describedly be reflected at 0 to 30 ℃ and for example carry out among DMF (dimethyl formamide) or the NMP (N-Methyl pyrrolidone) at polar non-solute.When described coupling reaction is finished, piperidines, potassium carbonate, DBU or other alkali well known to those skilled in the art joined in the described reaction (add or do not add other cosolvent) with the terminal Fmoc blocking group of effective removal.When described reaction is finished, add alcohol or can with the miscible solvent of water and/or water with the precipitation of peptides fragment, described fragments of peptides has SEQ ID NO:20 aminoacid sequence, and at C end amidatioon (H-AA (27-38)-NH ₂).

As Fig. 3 graphic extension, in liquid phase process, use fragments of peptides Fmoc-AA (27-37)-OH and leucyl amine (referring to Fig. 3, " H-Leu-NH ₂") combined preparation fragments of peptides H-AA (27-38)-NH ₂, with described fragments of peptides Fmoc-AA (27-37)-OH (571g, 205mmol, 1 equivalent), H-Leu-NH ₂(32.0g, 246mmol, 1.2 equivalents) and 6-Cl HOBT (41.7g, 246mmol, 1.2 equivalents) join among the DMF (4568ml, 8 volumes), with DIEA (53.6ml, 307.5mmol, 1.5 equivalents) handle and in stirring at room until dissolving (about 20 minutes).With described solution cooling, and add TBTU (79.0g, 246mmol, 1.2 equivalents).Be reflected at 0 ℃ of stirring with described, then 25 ℃ of stirrings.When the HPLC analysis shows that described reaction is finished, add piperidines (81ml, 820mmol, 4 equivalents) to remove described fragments of peptides Fmoc-AA (27-38)-NH ₂Fmoc blocking group (also can use other alkali, for example potassium carbonate, DBU etc.).To be reflected at 30 ℃ of stirrings, and show to react until HPLC and finish.Then described reactant mixture is cooled to below 5 ℃, and (8 volumes 4568mL) keep the gained slurry temperature to be lower than 10 ℃ slowly to add pre-cold water.Suspension was stirred 30 minutes, filter then and with 25% ethanol/water washing 2 times (2284mL, 4 volumes) at every turn.By in ethanol/water (adding or do not add diluted acid) and/or MTBE/ heptane or other similar solvent mixture again slurrying to remove residual piperidines and piperidines dibenzyl fulvene.As shown in Figure 3, product is isolating fragments of peptides H-AA (27-38)-NH ₂Preparation.

As Fig. 3 explanation, carry out liquid phase reactor, wherein fragments of peptides H-AA (27-38)-NH ₂(SEQ ID NO:20) and fragments of peptides Fmoc-AA (13-26)-OH (SEQ ID NO:18) combination, and go protection to produce fragments of peptides H-AA (13-38)-NH ₂(SEQ ID NO:35 has chemical group at N end and C end).

With fragments of peptides Fmoc-AA (13-26)-OH (460g, 167mmol, 1 equivalent), fragments of peptides H-AA (27-38)-NH ₂(460g, 172mmol, 1.03 equivalents) and 6-Cl HOBT (34g, 200mmol, 1.2 equivalents) join among the DMF (6900ml, 15 volumes), handle with DIEA (47ml, 267mmol .1.6 equivalent), and stir with the dissolving all solids.Gained solution is cooled to below 5 ℃.In reaction, add TBTU (64g, 200mmol, 1.2 equivalents), and will be reflected at 0 ℃ of stirring, then 25 ℃ of stirrings.In case HPLC analyzes the described reaction of demonstration and finishes, add piperidines (58ml, 668mmol, 4 equivalents) to remove Fmoc, stir described reaction then and finish until HPLC demonstration reaction.Solution is cooled to below 5 ℃, and slowly adds entry (6900mL, 15 volumes) with given pace and make temperature can not be raised to more than 10 ℃.After the gained suspension stirred 30 minutes, by solid collected by filtration and wash (2 times, each 2300mL, 5 volumes) and dry with water.By in ethanol/water (adding or do not add diluted acid) and/or MTBE/ heptane or other similar solvent mixture again slurrying remove residual piperidines and piperidines dibenzyl fulvene.By solid collected by filtration, washing, also dry to produce pure substantially white solid H-AA (13-38)-NH ₂(SEQ ID NO:35) determined by high performance liquid chromatography (HPLC) purity analysis.

As Fig. 3 explanation, fragments of peptides H-AA (13-38)-NH ₂(SEQ ID NO:35) in liquid phase reactor with fragments of peptides Ac-(1-12)-OH (SEQ ID NO:17) assembling with generation have SEQ ID NO:9 aminoacid sequence the HIV fusion inhibitor peptides (referring to, Fig. 3 for example, Ac-(1-38)-NH ₂).With fragments of peptides Ac-AA (1-12)-OH (130g, 58.5mmol, 1 equivalent) grind to form fine powder and with fragments of peptides H-AA (13-38)-NH ₂(303g, 58.5mmol, 1 equivalent) mixes.Described mixture is slowly joined 2: (20 volumes are 2600mL) and in the warm solution of DIEA (25.5mL, 146mmol, 2.5 equivalents) for 1DCM/DMF.Add HOAT (15.9g, 117mmol, 2.0 equivalents) and stir described mixture with the dissolving all solids.Gained solution is cooled to below 5 ℃, and adds TBTU (28.2g, 87.8mmol, 1.5 equivalents).Solution was stirred 30 minutes at 0 ℃,, finish until HPLC demonstration reaction then 25 ℃ of stirrings.Described solution is heated to 30-35 ℃, and add extra DCM (13 volumes, 1740mL) and water (1820mL, 14 volumes).Described mixture was stirred 5 minutes, allow layering then.Remove water layer and replace with fresh water (1820ml, 14 volumes).Layering repeats to amount to 5 times.Organic layer is distilled to original volume 1/3, and adds isopropyl alcohol (IPA; 1820ml, 14 volumes).Continue distillation to remove residual DC M.The gained slurry is cooled to below 5 ℃, and slowly adds entry (1820ml, 14 volumes).The solid shape that forms is collected by filtering, washes with water 2 times (each 520mL, 4 volumes), and dry to produce isolating HIV fusion inhibitor peptides Ac-AA (1-38)-NH ₂(SEQ ID NO:9) preparation is determined by the HPLC purity analysis.

As shown in Figure 3, can by removing side chain chemoproection group the de-protected method of peptide be removed HIV fusion inhibitor peptides Ac-AA (1-38)-NH by acidolysis or well known to those skilled in the art other ₂Side chain chemoproection group.In the present embodiment, HIV fusion inhibitor peptides Ac-AA (1-38)-NH ₂(60g is 8.1mmol) with TFA (trifluoroacetic acid): DDT (dithiothreitol, DTT): water (90: 10: 5; 570ml) handle, and stirring at room 6 hours.This solution is cooled to below 10 ℃, and (25 volumes 1500ml) make temperature keep below 10 ℃ slowly to add pre-cooling MTBE with given pace.The solid by filtration that generates is collected, with the MTBE washing, and dry.Then with the gained powder at acetonitrile (ACN; 10 volumes, 600mL) in slurrying, regulate pH to 4 between 5 with DIEA and acetic acid, so that described peptide decarboxylation.In case showing described reaction, finishes HPLC; pass through solid collected by filtration; wash with ACN; and drying is removed protection and decarboxylized peptide formulations with generation; described then peptide formulations has the isolating HIV fusion inhibitor peptides preparation of SEQ ID NO:9 aminoacid sequence by HPLC or other suitable chromatographic technique purification with generation.

Embodiment 6

Reference table 5 (group 3 and group 4) and Fig. 4 have illustrated and have used 2 particular peptide fragments (for example SEQ IDNO:29 and 30+Leu; Or SEQ ID NO:29 and 31) and use the synthetic method of fragment construction from part, described fragment construction from part to comprise 2 fragments of peptides are made up the HIV fusion inhibitor peptides that has SEQ ID NO:9 aminoacid sequence with generation by chemical bond (" assembling ") with HIV fusion inhibitor peptides of SEQ IDNO:9 aminoacid sequence.Physical property and dissolubility property that every kind of described fragments of peptides demonstrates make described fragments of peptides become the preferred peptide fragment, are used for using 2 fragments of peptides with high yield and the synthetic method with HIV fusion inhibitor peptides of SEQID NO:9 aminoacid sequence of high-purity.When selecting to be used for the fragments of peptides of two fragment assemble methods, the abutment of discovery between 2 assembled fragments (for example, have the fragments of peptides C end of SEQ IDNO:29 aminoacid sequence and have the fragments of peptides N end of SEQ ID NO:31 aminoacid sequence) have leucine and/or glutamic acid and help with high yield assembling and the purity level that obtains.

The fragments of peptides of 20 amino acids (" AA (1-20) ") before having SEQ ID NO:29 aminoacid sequence and comprise SEQ IDNO:9 by the standard solid-phase synthetic method being synthetic, the acetylation (" Ac " of described fragment N end, as chemical group), the C end has hydroxyl (OH) (referring to Fig. 6; Be also referred to as " Ac-AA (1-20)-OH " herein).By the synthetic fragments of peptides that has SEQ ID NO:30 aminoacid sequence and comprise 21-37 amino acids among the SEQ ID NO:9 (" AA (21-37) ") of standard solid-phase synthetic method, described fragment N end has Fmoc (as the chemoproection group), and the C end has-and OH is (referring to Fig. 6; Be also referred to as " Fmoc-AA (21-37)-OH " herein).

As table 5 group 3 and group 4 and shown in Figure 4, by the synthetic preparation of liquid phase fragments of peptides, wherein fragments of peptides Fmoc-AA (21-37)-OH chemical bond is to the fragments of peptides (comprising 21-38 amino acids among the SEQID NO:9) that has SEQ ID NO:31 aminoacid sequence in liquid phase on amidated SEQ ID NO:9 the 38th amino acids leucine with generation, and described fragments of peptides C holds amidatioon (as chemical group) (" Fmoc-AA (21-38)-NH ₂").In order in liquid phase process, to use fragments of peptides Fmoc-AA (21-37)-OH and leucine (" H-Leu-NH ₂") combined preparation fragments of peptides Fmoc-AA (21-38)-NH ₂, with fragments of peptides Fmoc-AA (21-37)-OH (30g, 7.43mmol, 1.0 equivalents), H-Leu-NH ₂* HCl (1.36g, 8.16mmol, 1.2 equivalents) and HOAT (1.52g, 11.2mmol, 1.5 equivalents) are dissolved among the DMF (450ml, 15 volumes), with DIEA (6.5ml, 37.3mmol, 5 equivalents) handle and in stirring at room until dissolving (about 30 minutes).This solution is cooled to 0 ± 5 ℃, and adds TBTU (2.86g, 8.91mmol, 1.2 equivalents), stirred 5 minutes, show to react 25 ± 5 ℃ of reactions 2 hours or until HPLC then and finish at 0 ± 5 ℃.

At isolated fragment H-AA (21-38)-NH ₂Remove fragments of peptides Fmoc-AA (21-38)-NH before ₂Fmoc chemoproection group.Add piperidines (7.3mL, 73.8mmol, 10 equivalents), and described solution was stirred 1 hour or analyzed basic all Fmoc of demonstration until HPLC and remove from described fragments of peptides at 25 ± 5 ℃.Cooling reactor adds entry (1000ml, 30 volumes), stirring free-pouring slurry 30 minutes below 10 ℃, passes through isolated by filtration then.The solid of collecting with the washing of 1: 1 ethanol/water, and in vacuum drying oven 35 ± 5 ℃ of dryings.Then with fragments of peptides slurrying 3 hours again in 1: 1 ethanol/water (450mL, 15 volumes).Collect and drying solid.The slurrying in 3: 1 normal hexane: MTBE (450mL, 15 volumes) of described then fragments of peptides is spent the night, and it is new dry to lay equal stress on by isolated by filtration then.As needs, described MTBE slurrying again can repeat to remove unnecessary piperidines.Products therefrom is isolating fragments of peptides H-AA (21-38)-NH ₂Preparation (referring to Fig. 4).

Carried out liquid phase reactor then, wherein with fragments of peptides H-AA (21-38)-NH ₂(SEQ ID NO:31) and fragments of peptides Ac-AA (1-20)-OH (SEQ ID NO:29) combination, with generation have SEQ ID NO:9 aminoacid sequence the HIV fusion inhibitor peptides (referring to Fig. 4, Ac-(1-38)-NH ₂).With fragments of peptides H-AA (21-38)-NH ₂(3.40g, 0.86mmol, 1 equivalent), fragments of peptides Ac-AA (1-20)-OH (3.00g, 0.86mmol, 1.0 equivalents) and HOAT (0.177g, 1.3mmol, 1.5 equivalent) and DIEA (0.599ml, 3.44mmol, 4 equivalents) be dissolved in DMAc (dimethyl acetylamide; 100ml, 33 volumes), be cooled to 0 ± 5 ℃.In reaction, add TBTU (0.331g, 1.03mmol, 1.2 equivalents).Be reflected at 0 ± 5 ℃ and stirred 5 minutes described, and stirred 3 hours or show that until HPLC described reaction finishes at 25 ± 5 ℃.Cooling reactor slowly adds entry (200ml, 66 volumes).The formation slurry was also stirring 30 minutes below 10 ℃ at least.Solid by filtration is separated and with other water washing.The solids of in vacuum drying oven, collecting 35 ± 5 ℃ of dryings.Products therefrom is HIV fusion inhibitor peptides Ac-AA that protect fully, isolating (1-38)-NH ₂(SEQ ID NO:9) preparation is determined by the HPLC purity analysis.Use the embodiment of the invention 5 described methods or well known to those skilled in the art other to go protection and decarboxylation method that described HIV fusion inhibitor peptides is removed protection (by removing side chain chemoproection group) and decarboxylation (at trp residue), purification (for example passing through HPLC) then then.Products therefrom is HIV fusion inhibitor peptides (in this explanation, N holds acetylation, and C holds amidatioon) preparation with SEQ ID NO:9 aminoacid sequence (goes protect and dehydroxylation).

Use similar technology and condition, use the get everything ready HIV fusion inhibitor peptides (referring to, table 4 and 5 for example) of SEQ ID NO:9 aminoacid sequence of other fragment assembling legal system that relates to the assembling of 2 fragments or 3 fragment assemblings.Should be understood that from this paper description the preferred peptide fragment that is used for by method preparation of the present invention has the HIV fusion inhibitor peptides of SEQ ID NO:9 aminoacid sequence can be used for getting rid of preferred peptide fragment fragments of peptides in addition.Similarly, be used for can be used for getting rid of preferred peptide slice groups fragments of peptides group in addition by the preferred peptide slice groups that method preparation of the present invention has the HIV fusion inhibitor peptides of SEQ ID NO:9 aminoacid sequence.

Embodiment 7

Another embodiment of the present invention relates to and can be used for synthetic method, fragments of peptides and fragments of peptides group with HIV fusion inhibitor peptides of SEQ ID NO:10 aminoacid sequence.It is evident that also that from this paper describes described method, fragments of peptides and fragments of peptides group can be used for synthetic HIV fusion inhibitor peptides with SEQ ID NO:10 aminoacid sequence, wherein said HIV fusion inhibitor peptides comprises one or more chemical groups:

Wherein one of amino terminal or carboxyl terminal or both are with chemical group (B, U, Z; Wherein B, U, Z can be identical chemical group or different chemical groups) to modify, described chemical group can include but not limited to one or more following groups: reactive functional groups, chemoproection group (CPG) and joint.Relate to and prepare when having the HIV fusion inhibitor peptides of SEQ ID NO:10 aminoacid sequence; the illustrative examples of fragments of peptides, fragments of peptides group and protected fragments of peptides (fragments of peptides with one or more chemical groups) includes but not limited to, the listed fragments of peptides of difference in the table 7,8 and 9.

Table 7

??SEQ??ID??NO：	Aminoacid sequence	Amino acid sites in SEQ ID NO:10
			??17	??TTWEAWDRAIAE	??1-12
??41	??YAARIEALLRAAQE	??13-26
			??42	??QQEKLEAALRE	??27-37
??43	??QQEKLEAALREL	??27-38
			??21	??TTWEAWDRAIA	??1-11
??44	??EYAARIEALLRAAQE	??12-26
			??23	??TTWEAWDRAI	??1-10
??45	??AEYAARIEALLRAAQE	??11-26
			??25	??TTWEAWDRA	??1-9
??46	??IAEYAARIEALLRAAQE	??10-26
			??27	??TTWEAWDR	??1-8
??47	??AIAEYAARIEALLRAAQE	??9-26
			??29	??TTWEAWDRAIAEYAARIEAL	??1-20
??48	??LRAAQEQQEKLEAALRE	??21-37
			??49	??LRAAQEQQEKLEAALREL	??21-38
??32	??TTWEAWDRAIAEYAARIE	??1-18
			??50	??ALLRAAQEQQEKLEAALRE	??19-37
??51	??ALLRAAQEQQEKLEAALREL	??19-38
			??52	??YAARIEALLRAAQEQQEKLEAALREL	??13-38
??53	??EYAARIEALLRAAQEQQEKLEAALREL	??12-38
			??54	??AEYAARIEALLRAAQEQQEKLEAALREL	??11-38
??55	??IAEYAARIEALLRAAQEQQEKLEAALREL	??10-38
			??56	??AIAEYAARIEALLRAAQEQQEKLEAALREL	??9-38
??57	??WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF
			??58	??YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNIT
??59	??YQEWERKVDFLEENITALLEEAQIQQEKNMYELQKL

The present invention further provides specific fragments of peptides group, described fragments of peptides group in the method for synthetic HIV fusion inhibitor peptides with SEQ IDNO:10 aminoacid sequence as intermediate.Fragments of peptides group provided by the invention comprises the group 1-14 shown in the table 8 (numbering of group only for convenience of description).Some fragments of peptides group can be used for getting rid of other fragments of peptides group.

Table 8

Group number	Fragments of peptides	Amino acid sites in SEQ ID NO:10
			??1	??TTWEAWDRAIAE(SEQ?ID?NO：17)??YAARIEALLRAAQE(SEQ?ID?NO：41)??QQEKLEAALRE(SEQ?ID?NO：42)	??1-12??13-26??27-37
??2	??TTWEAWDRAIAE(SEQ?ID?NO：17)??YAARIEALLRAAQE(SEQ?ID?NO：41)??QQEKLEAALREL(SEQ?ID?NO：43)	??1-12??13-26??27-38
			??3	??TTWEAWDRAIAEYAARIEAL(SEQ?ID?NO：29)??LRAAQEQQEKLEAALRE(SEQ?ID?NO：48)	??1-20??21-37
??4	??TTWEAWDRAIAEYAARIEAL(SEQ?ID?NO：29)??LRAAQEQQEKLEAALREL(SEQ?ID?NO：49)	??1-20??21-38
			??5	??TTWEAWDRAIA(SEQ?ID?NO：21)??EYAARIEALLRAAQE(SEQ?ID?NO：44)??QQEKLEAALRE(SEQ?ID?NO：42)	??1-11??12-26??27-37
??6	??TTWEAWDRAI(SEQ?ID?NO：23)??AEYAARIEALLRAAQE(SEQ?ID?NO：45)??QQEKLEAALRE(SEQ?ID?NO：42)	??1-10??11-26??27-37
			??7	??TTWEAWDRA(SEQ?ID?NO：25)??IAEYAARIEALLRAAQE(SEQ?ID?NO：46)??QQEKLEAALRE(SEQ?ID?NO：42)	??1-9??10-26??27-37
??8	??TTWEAWDR(SEQ?ID?NO：27)??AIAEYAARIEALLRAAQE(SEQ?ID?NO：47)??QQEKLEAALRE(SEQ?ID?NO：43)	??1-8??9-6??27-37
			??9	??TTWEAWDRAIA(SEQ?ID?NO：21)??EYAARIEALLRAAQE(SEQ?ID?NO：44)??QQEKLEAALREL(SEQ?ID?NO：43)	??1-11??12-26??27-38
??10	??TTWEAWDRAI(SEQ?ID?NO：23)??AEYAARIEALLRAAQE(SEQ?ID?NO：45)??QQEKLEAALREL(SEQ?ID?NO：43)	??1-10??11-26??27-38
			??11	??TTWEAWDRA(SEQ?ID?NO：25)??IAEYAARIEALLRAAQE(SEQ?ID?NO：46)??QQEKLEAALREL(SEQ?ID?NO：43)	??1-9??10-26??27-38
??12	??TTWEAWDR(SEQ?ID?NO：27)??AIAEYAARIEALLRAAQE(SEQ?ID?NO：47)??QQEKLEAALREL(SEQ?ID?NO：43)	??1-8??9-26??27-38
			??13	??TWEAWDRAIAEYAARIE(SEQ?ID?NO：32)??ALLRAAQEQQEKLEAALRE(SEQ?ID?NO：50)	??1-18??19-37
??14	??TTWEAWDRAIAEYAARIE(SEQ?ID?NO：32)??ALLRAAQEQQEKLEAALREL(SEQ?ID?NO：51)	??1-18??19-38

Table 9

??SEQ??ID?NO：	Aminoacid sequence	Amino acid sites in SEQ ID NO:10
			??17	??Ac-TTWEAWDRAIAE	??1-12
??41	??CPG-YAARIEALLRAAQE	??13-26
			??42	??CPG-QQEKLEAALRE	??27-37
??42	??CPG-QQEKLEAALRE??I??IvDde	??27-37
			??43	??QQEKLEAALREL-NH 2	??27-38
??29	??Ac-TTWEAWDRAIAEYAARIEAL	??1-20
			??48	??CPG-LRAAQEQQEKLEAALRE	??21-37
??49	??LRAAQEQQEKLEAALREL-NH 2	??21-38
			??21	??Ac-TTWEAWDRAIA	??1-11
??44	??CPG-EYAARIEALLRAAQE	??12-26
			??23	??Ac-TTWEAWDRAI	??1-10
??45	??CPG-AEYAARIEALLRAAQE	??11-26
			??25	??Ac-TTWEAWDRA	??1-9
??46	??CPG-IAEYAARIEALLRALQE	??10-26
			??27	??Ac-TTWEAWDR	??1-8
??47	??CPG-AIAEYAARIEALLRAAQE	??9-26
			??32	??Ac-TTWEAWDRAIAEYAARIE	??1-18
??50	??CPG-ALLRAAQEQQEKLEAALRE	??19-37
			??51	??ALLRAAQEQQEKLEAALREL-NH 2	??19-38

Reference table 8 (group 3 and group 4) has illustrated and has used 2 particular peptide fragments (for example SEQ ID NO:29 and 48+Leu; Or SEQ ID NO:29 and 49) and use the synthetic method of fragment construction from part, described fragment construction from part to comprise and make up 2 fragments of peptides have SEQ ID NO:10 aminoacid sequence with preparation HIV fusion inhibitor peptides by chemical bond (" assembling ") with HIV fusion inhibitor peptides of SEQ IDNO:10 aminoacid sequence.In order in liquid phase process, to use fragments of peptides (" Fmoc-AA (21-37)-OH ") and leucine (" H-Leu-NH with SEQ ID NO:48 ₂") combined preparation has the fragments of peptides (" Fmoc-AA (21-38)-NH of SEQ ID NO:49 ₂"), with fragments of peptides Fmoc-AA (21-37)-OH (30.01g, 7.98mmol, 1.0 equivalents), H-Leu-NH ₂* HCl (1.48g, 8.78mmol, 1.1 equivalents) and HOAT (1.63g, 11.97mmol, 1.5 equivalents) are dissolved among the DMF (450ml, 15 volumes), with DIEA (7.0ml, 39.91mmol, 5 equivalents) handle and in stirring at room until dissolving (about 30 minutes).Described solution is cooled to 0 ± 5 ℃, and adds TBTU (3.09g, 9.58mmol, 1.2 equivalents), stirred 5 minutes, show that 25 ± 5 ℃ of reactions 2 hours or until HPLC described reaction finishes then at 0 ± 5 ℃.

At isolated fragment H-AA (21-38)-NH ₂Remove fragments of peptides Fmoc-AA (21-38)-NH before ₂Fmoc chemoproection group.Add piperidines (8.0ml, 79.8mmol, 10 equivalents), and described solution was stirred 1.5 hours or analyzed basic all Fmoc of demonstration until HPLC and all removed from described fragments of peptides at 25 ± 5 ℃.With reactor cooled, add entry (1000ml, 30 volumes), stirring free-pouring suspension 30 minutes below 10 ℃, pass through isolated by filtration then.The solid of collecting washs with 1: 3 ethanol/water, and in vacuum drying oven in 35 ± 5 ℃ of dryings.Then with fragments of peptides slurrying 3 hours again in 1: 3 ethanol/water (400mL, 13 volumes).Collect and drying solid, then the slurrying in 3: 1 normal hexane: MTBE (400mL, 13 volumes) of described fragments of peptides is spent the night, it is new dry to lay equal stress on by isolated by filtration.As needs, described MTBE slurrying again can repeat to remove unnecessary piperidines.Products therefrom is isolating fragments of peptides H-AA (21-38)-NH ₂Preparation (referring to table 9, SEQ ID NO:49).

Carry out liquid phase reactor then, wherein fragments of peptides H-AA (21-38)-NH ₂(SEQ ID NO:49) and fragments of peptides Ac-AA (1-20)-OH (SEQ ID NO:29, table 9) combination have the HIV fusion inhibitor peptides of SEQ ID NO:10 aminoacid sequence (referring to, Ac-(1-38)-NH for example with generation ₂).With fragments of peptides H-AA (21-38)-NH ₂(3.14g, 0.86mmol, 1 equivalent), fragments of peptides Ac-AA (1-20)-OH (3.00g, 0.86mmol, 1.0 equivalent) and HOAT (0.18g, 1.3mmol, 1.5 equivalents) and DIEA (0.599ml, 3.44mmol, 4 equivalents) be dissolved in DMAc (100ml, 33 volumes), be cooled to 0 ± 5 ℃.In reaction, add TBTU (0.331g, 1.03mmol, 1.2 equivalents).Be reflected at 0 ± 5 ℃ and stirred 5 minutes described, and stirred 3 hours or show that until HPLC described reaction finishes at 25 ± 5 ℃.Cooling reactor slowly adds entry (250ml, 83 volumes).Slurry forms and was stirring at least 30 minutes below 10 ℃.Solid by filtration is separated and with extra water washing.The solid of collecting in vacuum drying oven in 35 ± 5 ℃ of dryings.Products therefrom is HIV fusion inhibitor peptides Ac-AA that protect fully, isolating (1-38)-NH ₂(SEQ ID NO:10) preparation is determined by the HPLC purity analysis.Use the embodiment of the invention 4 described methods or well known to those skilled in the art other to go protection and decarboxylation method then to described HIV fusion inhibitor peptides Ac-AA (1-38)-NH ₂Go protection (by removing side chain chemoproection group) and decarboxylation (at trp residue).Behind the purification, products therefrom is HIV fusion inhibitor peptides (N holds acetylation, and C the holds amidatioon) preparation (go protect and dehydroxylation) of the isolating SEQ of having ID NO:10 aminoacid sequence, is determined by HPLC.

Use similar technology and condition, use the get everything ready HIV fusion inhibitor peptides (referring to, table 8 and 9 for example) of SEQ ID NO:10 aminoacid sequence of other fragment assembling legal system that relates to the assembling of 2 fragments or 3 fragment assemblings.Should be understood that from this paper description the fragments of peptides that is used for by method preparation provided by the invention has the HIV fusion inhibitor peptides of SEQ IDNO:10 aminoacid sequence can be used for getting rid of other fragments of peptides.Similarly, be used for can be used for getting rid of other fragments of peptides group by the fragments of peptides group that method preparation provided by the invention has the HIV fusion inhibitor peptides of SEQ ID NO:10 aminoacid sequence.

Embodiment 8

The present invention further provides and used antiviral peptide for example HIV fusion inhibitor peptides (this peptide itself or as the active drug substance in the compositions provided by the invention) treatment HIV infection and/or AIDS or infect and/or the method for the part of AIDS therapeutic scheme as HIV.The antiviral activity of HIV fusion inhibitor peptides can be used for suppressing the method that HIV transmits to target cell, described method comprises makes virus and/or cell and certain amount of H IV fusion inhibitor peptides, described amount effectively suppresses cell HIV and infects, more preferably, effectively suppress the virus of HIV mediation and the fusion of target cell.Described method can be used for treating HIV infected patient (treatment with) or treatment just has been exposed to or have excessive risk to be exposed to patient's (prevention usefulness) of (for example, by medication or high risk behavior) HIV.Therefore, for example, in the example of HIV-1 infected patient, the HIV fusion inhibitor peptides of effective dose is to be enough to the dosage that (combining by this peptide itself and/or with the multiple dose scheme) reduces the interior HIV virus load of institute's patient's body for the treatment of.Know as those skilled in the art, the standard method of multiple mensuration HIV virus load is arranged, include but not limited to, measure by the peripheral blood lymphocytes quantitative culture with by blood plasma HIV RNA.But described HIV fusion inhibitor peptides single ground, off and on, use termly or continuously, method of application is determined by the medical worker, for example by the monitoring virus load.According to the particular composition that comprises the HIV fusion inhibitor peptides provided by the invention, and whether further comprising factors such as pharmaceutical acceptable carrier and/or macromolecular carrier such as particular composition provided by the invention, the HIV fusion inhibitor peptides can or may be used with a couple of days to several weeks in the longer cycle.In addition, the HIV fusion inhibitor peptides in antiviral therapy can with other be used for the treatment of the antiviral drugs of HIV or prevention medicament be used in combination or be used for the treatment of scheme (for example, use simultaneously or alternately and a kind of medicine and another kind of drug regimen) in.

A kind of treatment that relates to the antiviral agent combination commonly used is called as HAART (high activity antiretroviral therapy).HAART typically makes up 3 kinds or more kinds of medicine with anti-HIV antiviral activity, and typically relates to the medicine (" class " relates to the virus protein or the process of mechanism of action or drug targeting) more than a class.Therefore, the compositions of the HIV of comprising fusion inhibitor peptides provided by the invention can be used (for example as monotherapy) separately or use in therapeutic scheme or use jointly, described use jointly to relate to be used for the treatment of that HIV infects and/or the combination of other therapeutic agent of AIDS, describe in detail as this paper.

For example, in one embodiment, one or more therapeutic agents can make up with the HIV fusion inhibitor peptides in compositions provided by the invention in treatment.These combinations can comprise at least a antiviral agent except that described HIV fusion inhibitor peptides.These combinations can, for example the antiviral agent (being used for the treatment of HIV infects) by effective dose present approval or that ratify in the future prepares, described antiviral agent includes but not limited to be selected from one or more other therapeutic agents of following medicament: antiviral agent is cytokine for example, for example rIFN α, rIFN β, rIFN γ; Reverse transcriptase inhibitors, include but not limited to Abacavir, AZT (zidovudine), ddC (zalcitabine), nevirapine, ddl (Didanosine), FTC (emtricitabine), (+) and (-) FTC, reverset, 3TC (lamivudine), GS 840, GW-1592, GW-8248, GW-5634, HBY097, dilazep Wei Ding, efavirenz, d4T (stavudine), FLT, TMC125, adefovirdipivoxil, Tai Luofuwei and alovudine; Protease inhibitor, include but not limited to amprenavir, CGP-73547, CGP-61755, DMP-450, indinavir, nelfinavir, PNU-140690, ritonavir, Saquinavir, Compound E, tipranavir, atazanavir, Lopinavir, ABT378, ABT538 and MK639; Virus mRNA adds medicated cap inhibitor, for example ribavirin; Amphotericin B as lipid binding molecule with HIV (human immunodeficiency virus)-resistant activity; Castanospermine as glycoprotein processing inhibitor; Viral entry inhibitor, for example if fusion inhibitor (En Fuwei ground, T1249, other fusion inhibitor peptides, and micromolecule), SCH-D, UK-427857 (Pfizer), TNX-355 (Tanox Inc.), AMD-070 (AnorMED), Pro140, Pro 542 (Progenies), FP-21399 (EMD Lexigen), BMS806, BMS-488043 (Bristol-Myers Squibb), Malawi are (UK-427857), ONO-4128, GW-873140, AMD-887, CMPD-167 and GSK-873,140 (GlaxoSmithKline); CXCR4 antagonist, for example AMD-070; Lipid and/or cholesterol interaction regulator, for example procaine hydrochloride (SP-01 and SP-01A); Integrase inhibitor includes but not limited to, L-870 and 810; RNA enzyme H inhibitor; Rev or REV inhibitor; The vif inhibitor (for example come from the proline rich of vif peptide, come from the peptide of HIV-1 protease N end); Virus processing inhibitor includes but not limited to betulinol and dihydro betulin derivatives (for example PA-457); And immunomodulator, include but not limited to AS-101, granulocyte macrophage colony stimulating factor, IL-2, valproic acid and Thymopentin.Understand as HIV infection and/or AIDS treatment those skilled in the art, the composition of medicine treatment can comprise the therapeutic agent that two or more have same function mechanism, maybe can comprise the therapeutic agent that two or more have different mechanism of action.

The effective dose of described exemplary other therapeutic agent that can use with HIV fusion inhibitor peptides provided by the invention and/or combination of compositions is known in the art.And the effective dose of using of HIV fusion inhibitor peptides provided by the invention or pharmaceutical composition can be definite by method well known to those skilled in the art, for example determines by definite effectiveness, biological half-life, bioavailability and toxicity.In one embodiment, those skilled in the art use data in the external and body of routine well known to those skilled in the art to determine the effective dose and the dosage range of HIV fusion inhibitor peptides.For example, the Infection in Vitro analysis of all antiviral activities as described in the present invention makes those skilled in the art can determine that chemical compound (making up as independent active component or with other active component) suppresses viral infection (for example 50% inhibition, the IC of preset range ₅₀Or 90% suppress IC ₉₀) or suppress the needed mean inhibitory concentration of virus replication (IC).Those skilled in the art can use the pharmacokinetic data of one or more master patterns to select proper dosage then, obtain equaling or exceeding the minimum plasma concentration (C of the described active component of the predetermined value that suppresses viral infection or virus replication thus _[min]).Though dosage range typically depends on the route of administration and the dosage formulation of selection, when using, for example route of administration includes but not limited to, subcutaneous, parenteral, Intradermal or oral can be from about 1mg/kg body weight to about 100mg/kg body weight as the exemplary dose scope of the chemical compound of active component; And more preferably from being not less than the 1mg/kg body weight to being no more than the 10mg/kg body weight.In one embodiment, use by injection (using for example subcutaneous injection).In one embodiment, use the HIV fusion inhibitor peptides.

Therefore, provide and suppressed the method that HIV transmits to cell, described method comprises uses compositions of the present invention, and described compositions comprises the HIV fusion inhibitor peptides of the effective dose that suppresses cell HIV infection.Described method can further comprise and is used for the treatment of to patient's combined administration compositions of the present invention and other that HIV infects and/or the therapeutic agent of AIDS, described method is the combination (simultaneously or successively, or the part of therapeutic scheme) of using the therapeutic agent of the HIV fusion inhibitor peptides provided by the invention that comprises effective dose or pharmaceutical composition to individuality.Also provide and suppressed the method that HIV enters, described method comprises to the patient that these needs are arranged uses compositions of the present invention, and described compositions comprises and suppresses the HIV fusion inhibitor peptides that virus enters the effective dose of target cell.Described method can further comprise combined administration compositions of the present invention and effective dose one or more can be used for treating other inhibitor that HIV infects, for example viral entry inhibitor.

Embodiment 9

Prepare method for compositions provided by the invention as detailed below.In addition, exemplary composition has been described.

Material: Sucrose acetoisobutyrate (SAIB) is from Eastman Chemicals.Polylactide (PLA) and polylactide-co-glycolide copolymer (PLGA) are from Lakeshore Biomateials.The difference of PLA and PLGA is lactide: Acetic acid, hydroxy-, bimol. cyclic ester ratio, molecular weight and their end group.All PLGA that use in this research are 50: 50 lactides: Acetic acid, hydroxy-, bimol. cyclic ester.Molecular weight is with the digital classification in the title.The molecular weight valuation is 10000 times of described numeral.End group is carboxylic acid (A), methyl ester (M) or Laurel alcohol ester (L).N-N-methyl-2-2-pyrrolidone N-(NMP) is from Spectrum.Benzyl benzoate and glyceryl triacetate are from Sigma.Guanidine hydrochloride is from Amresco.Tris-HCl is from Sigma.4-(2-pyridylazo) resorcinol is from Sigma.Methanol is from VWR.Zinc sulphate heptahydrate is from Sigma.Zinc chloride is from Sigma.

T1144 fret peptide preparation: prepare the T1144 fret peptide according to following rules.

Spray drying: with the T1144 peptide at pH less than 4 or be dissolved in the water usually greater than 6 o'clock.Use 1NNaOH or 1N HCl to regulate pH.Peptide solution is sprayed onto in the heating cabinet by spray nozzle.Artificially collect exsiccant peptide particles.

Peptide can further prepare by above-mentioned spray drying process, but adds excipient to spray drying soln, thus excipient is combined with peptide.

Salt precipitation or pH precipitation: with peptide at pH less than 4 or be dissolved in the water usually greater than 6 times.Use 1N NaOH or 1N HCl to regulate pH.Add saline solution or strong acid/alkali to cause precipitation.Precipitate by centrifugal collection, lyophilization drying, and cross 200 μ m sieve with the control granular size.

Carrier preparation: prepare carrier according to following method.

SAIB carrier preparation: can reach the appropriate amount SAIB heating of required final concentration and join among the NMP mix homogeneously.

The preparation of SAIB/PLA carrier: the appropriate amount PLA that can reach required final concentration is dissolved in NMP, benzyl benzoate or glyceryl triacetate.The appropriate amount SAIB heating of required final concentration be can reach and described PLA solution, mix homogeneously joined.

PLA, PLG and the preparation of PLGA carrier: appropriate amount PLA, PLG and the PLGA that can reach required PLA, PLG or PLGA final concentration are dissolved in NMP.

Can prepare compositions by any method well known to those skilled in the art.In the present embodiment, fret peptide (sedimentary or spray-dired) is joined in the bottle.Carrier is joined in the described bottle, and contents mixed is even.In some cases, need be heated to about 40 ℃ to guarantee correct mixing.Prescription quantitatively is peptide weight/prescription (representing with mg/g).

By the mode similar, absorb definite peptide content according to tryptophan and tyrosine to the Edelhoch method.Briefly, about 1mg fret peptide is dissolved in the 1mL 8M guanidine hydrochloride.276,280 and 288nm measure the uv absorption of solution.Use the absorption value of these mensuration, and the quantity and the peptide molecular weight of tryptophan and tyrosine residue in the known example weight, sample volume, peptide, determine the peptide content (%w/w) in the solid.

Use the ultraviolet absorption analysis to determine metal cation content, 4-(2-pyridylazo) resorcinol (PAR) is used in described analysis, and PAR is known and M ²⁺Form a kind of metallochromic indicator of 2: 1 complex.Briefly, about 1mg solid being dissolved in 1mL comprises in the Tris-HCl buffer (pH 8) of 6M guanidine hydrochloride.Diluting described solution (using identical buffer) makes the metal cation final concentration be 1-10 μ M.50 μ l 0.1M PAR are joined in the 950 μ L dilute solutions.After the balance, the ultraviolet of measuring test solution at 500nm absorbs.Based on the linear least-squares analytical calculation metal cation concentration of the standard curve that obtains on the same day, and the metal cation content (wt%) of definite sample.

Analyze the peptide concentration of determining in the blood plasma by LC-MS.Use the dilution in acetonitrile plasma sample that comprises 0.5% (v/v) formic acid of 3 times of volumes, centrifugal, and the direct analysis supernatant.The use gradient elution (the 10mM ammonium acetate, pH6.8: acetonitrile, 0.6mL/min) carry out chromatography, move 6 minutes altogether.Separate on PhenomenexLuna C8 (2) 50 * 2mm posts, described post is protected by 4 * 2mm PhenomenexSecurityGuard C8 guard column.On Sciex API4000 or API4000 Qtrap equipment, carry out mass spectral analysis (ESI+), use list-quadrupole pattern usually, detect [M+3H] ³⁺Or [M+4H] ⁴⁺Ion.Make up the TRI-1144 calibration curve from 30ng/mL to 30 μ g/mL.The result is expressed as time dependent plasma peptides concentration.Use following abbreviation: C _Max=maximum plasma peptides concentration; t _Max=C _MaxTime; t _0.1=plasma peptides lowering of concentration is to the time below the 0.1 μ g/ml; And t _0.01For standardization plasma peptides lowering of concentration to the time below the 0.01 μ g/ml.In some cases, for ease of comparing, plasma concentration is standardized as 3mg peptide/kilogram the weight of animals.

The animals administer mode is as follows.The compositions that comprises excessive T1144 is inhaled into the 1cc syringe by the 16G syringe needle.Replace described syringe needle with 18G or 21G syringe needle, and syringe empties to correct dose.Subcutaneous area administration between the animal scapula.Rat (400g) administration 400 μ L, 3 animals of each dosage group.Macaque (2.5kg) administration 400 μ L or 1000 μ L, every group of 3 animals.

All pharmacokinetic datas are collected from rat or monkey.In some cases, for ease of comparing, plasma concentration is standardized as 3mg peptide/kilogram animal.

The compositions that comprises peptide below having prepared.

T1144/ zinc precipitate A. T1144 is water-soluble.Regulate pH to about 6.2 and to add water to concentration be 25mg/mL.Described solution is passed through 0.22 μ m filter.With 2mL 0.1M ZnSO ₄Join in the 80mLT1144 solution.The gained suspension is centrifugal, abandon supernatant, and freezing precipitation.With described precipitation lyophilizing and by 200 μ m sieve.

T1144/ zinc deposit B. T1144 is water-soluble.Regulate pH to about 6.2 and to add water to concentration be 25mg/mL.Described solution is passed through 0.22 μ m filter.With 60mL 0.1M ZnSO ₄Join in the 120mLT1144 solution.The gained suspension is centrifugal, abandon supernatant, and freezing precipitation.With described precipitation lyophilizing and by 200 μ m sieve.

T1144/ zinc deposit C. T1144 is water-soluble.Regulate pH to about 5.7 and to add water to concentration be 40mg/mL.Described solution is passed through 0.22 μ m filter.General＜100mg ZnCl ₂Join in the 20mL T1144 solution.The gained suspension is centrifugal, abandon supernatant, and precipitate with the 1mL water washing.Gained precipitated freezing, lyophilizing and by 200 μ m sieve.

T1144/ zinc deposit D. with 5mL water washing deposit B, centrifugal and abandon supernatant.This step repeats 2 times again.Gained precipitated freezing, lyophilizing and by 200 μ m sieve.

T1144/ zinc deposit E. with 445mg ZnSO ₄ ^*7H ₂O is dissolved in 2mL water.With the slurrying in zinc solution of 1.0g deposit D.Slurry is freezing, lyophilizing is also sieved by 200 μ m.

F. is water-soluble with T1144 for T1144/ zinc precipitation.Regulate pH to about 6.2 and to add water to concentration be 25mg/mL.Described solution is passed through 0.22 μ m filter.Add 5mL 1N acetic acid, reduce pH to about 5.The gained suspension is centrifugal, abandon supernatant, and freezing precipitation.To precipitate lyophilizing and pass through 200 μ m sieve.

G. is with 230mg ZnSO for T1144/ zinc precipitation ₄ ^*7H ₂O is dissolved in 2mL water.500mg is precipitated F slurrying in zinc solution.Slurry is freezing, lyophilizing is also sieved by 200 μ m.

H. is water-soluble with T1144 for T1144/ zinc precipitation.Regulate pH to about 8.4 and to add water to concentration be 50mg/mL.Described solution is passed through 0.22 μ m filter.Adding methanol to concentration is 25mg/mL (50: 50 methanol: water).Will about 1mL 0.1M ZnSO ₄Join in the 20mL TRI-1144 solution.The gained suspension is centrifugal, abandon supernatant, and freezing precipitation.To precipitate lyophilizing and pass through 200 μ m sieve.

I. is water-soluble with TRI-1144 for T1144/ zinc precipitation.Regulate pH to about 8.4 and to add water to concentration be 50mg/mL.Described solution is passed through 0.22 μ m filter.Adding methanol to concentration is 25mg/mL (50: 50 methanol: water).Regulate pH to about 5.The gained suspension is centrifugal, abandon supernatant, and freezing precipitation.To precipitate lyophilizing and pass through 200 μ m sieve.

J. is water-soluble with T1144 for T1144/ zinc precipitation.Regulate pH to about 6.2 and to add water to concentration be 25mg/mL.Described solution is passed through 0.22 μ m filter.With 60mL 0.1M ZnSO ₄Join in the 120mLT1144 solution.The gained suspension is centrifugal, abandon supernatant, and freezing precipitation.To precipitate lyophilizing and pass through 150 μ m sieve.

T1144/ zinc precipitate K. T1144 is water-soluble.Regulate pH to about 6.2 and to add water to concentration be 25mg/mL.Described solution is passed through 0.22 μ m filter.With 1mL 0.1M ZnSO ₄Join in the 40mLT1144 solution.The gained suspension is centrifugal, abandon supernatant, and freezing precipitation.To precipitate lyophilizing and pass through 150 μ m sieve.

T1144/ zinc precipitation L. is with 30mL water washing 1.0g precipitation J, and is centrifugal and abandon supernatant.This step repeats 1 time again.Gained precipitated freezing, lyophilizing and by 200 μ m sieve.

M. is water-soluble with T1144 for T1144/ zinc precipitation.Regulate pH to about 6.3 and to add water to concentration be 25mg/mL.Described solution is passed through 0.22 μ m filter.25mL T1144 solution is sprayed onto the violent blended 0.3M ZnSO of 50mL by spray nozzle (mode is similar to spray drying) ₄In the solution.The gained suspension is centrifugal, abandon supernatant, and freezing precipitation.To precipitate lyophilizing and pass through 150 μ m sieve.

N. is water-soluble with T1144 for T1144/ zinc precipitation.Regulate pH to about 6.3 and to add water to concentration be 50mg/mL.Adding methanol to T1144 solution final concentration is 25mg/mL.Described solution is passed through 0.22 μ m filter.25mL T1144 solution is sprayed onto the violent blended 0.1M ZnSO of 50mL by spray nozzle (mode is similar to spray drying) ₄In the solution.The gained suspension is centrifugal and abandon supernatant.Precipitate 3 times with the 10mL water washing.Suspension is centrifugal, abandon supernatant and freezing precipitation.To precipitate lyophilizing and pass through 200 μ m sieve.

Table 9: fret peptide compositions

Fret peptide	Peptide content (%)	Zinc content (%)
			??PPT?A	??90.6	??1.6
??PPT?B	??72.5	??7.8
			??PPT?C	??87.7	??1-5
??PPT?D	??89.3，87.9	??2.9，2.4
			??PPT?E	??70.1	??10.0
??PPT?F	??88.3	??N/A
			??PPT?G	??71.7	??7.7
??PPT?H	??90.6，89.7	??1.7，1.9
			??PPT?I	??87.7	??N/A
??PPT?J	??60.2	??11.5
			??PPT?K	??88.2	??1.7
??PPT?L	??93.7	??1.9
			??PPT?M	??60.9	??12.4
??PPT?N	??92.5	??2.1

In the following manner above-mentioned composition is applied to rat or monkey.The outcome expectancy that obtains in rat or monkey rationally is associated with people's result.

Deposit D is at 74: 11: 15 SAIB: PLA3L: be formulated as 100mg/g among the NMP, and to macaque administration 1000 μ L.As Fig. 5 (--◆--) shown in, plasma concentration continues to be higher than in 12 days the desired value of 1 μ g/mL, has surpassed 7 days object time.Precipitation J was at 40: 60 PLA3: be formulated as 50mg/g among the NMP, and to macaque administration 400 μ L.Shown in Fig. 5 (--■--), plasma concentration continues to be higher than in 7 days the desired value of 1 μ g/mL; More heavy dose of, for example 1000 μ L 100mg/g precipitation J may continue 10-12 days generation target plasma concentration.This show T1144 can be in SAIB/PLA or PLA carrier subcutaneous delivery, and continue to surpass the plasma concentration that provides in a week above desired value (i.e. 1 μ g/mL).

Deposit D is at 74: 11: 15 SAIB: PLA3L: be formulated as 100mg/g among the NMP, and to rat administration 400 μ L.As Fig. 6 (--◆--) shown in, plasma concentration continues to be higher than in 6 days the desired value of 1 μ g/mL, near meeting 7 days object time.Precipitation J was at 40: 60 PLA3: be formulated as 50mg/g among the NMP, and to rat administration 400 μ L.Shown in Fig. 6 (--■--), plasma concentration continues to be higher than in 7 days the desired value of 1 μ g/mL.This shows that described preparation provides similar T1144 to continue to send in rodent and Primate model.

SAIB: the PLA ratio is to the influence of rat pharmacokinetic property. with precipitate A at SAIB: PLA3M: be formulated as 50mg/g in the NMP carrier and to rat administration 400 μ l.As shown in Figure 7, reduce SAIB: PLA ratio (being more PLA) can reduce C _Max, increase t _MaxAnd increase t _0.01With deposit B also at SAIB: PLA3M: be formulated as 50mg/g in the NMP carrier and to rat administration 400 μ l.Shown in Fig. 8 and 9, the result is similar in nature to the result that precipitate A presents.But SAIB: the PLA ratio influences the degree that deposit B sends and is lower than the influence that it is sent precipitate A.This shows and reduces SAIB: the PLA ratio can improve T1144 continue send, and precipitation character, especially the zinc content in the precipitation can influence delivery rate.

Substrate: the solvent ratio is to the influence of rat pharmacokinetic property. with deposit B at SAIB: PLA3M: be formulated as 50mg/g in the NMP carrier and to rat administration 400 μ l.As shown in figure 10, with respect to lower PLA level, about 10% PLA level can be slowed down delivery of peptides.

Type of solvent is to the influence of rat pharmacokinetic property. with the SAIB of deposit B at 75: 5: 20: PLA3M: be formulated as 50mg/g in solvent (being glyceryl triacetate, benzyl benzoate or the NMP) carrier and to rat administration 400 μ l.As shown in figure 11, because type of solvent changes C _MaxReduction, t _MaxIncrease and t _0.01Increase.NMP can provide the pharmacokinetic property more better than glyceryl triacetate, and glyceryl triacetate is better than the benzyl benzoate performance.This shows that type of solvent can influence delivery of peptides.

Peptide concentration is to the influence of rat pharmacokinetic property. with the SAIB of deposit B at 75: 5: 20: PLA3M: preparation and to rat administration 400 μ l in the NMP carrier.The result and shows that as shown in figure 12 in this carrier, improving peptide concentration does not have adverse effect to delivery of peptides.

Volume injected is to the influence of rat pharmacokinetic property. with the SAIB of deposit B at 74: 11: 15: PLA3M: be formulated as 100mg/g in the NMP carrier and to the rat administration.As shown in figure 13, dose volume has no significant effect continuing delivery parameter; But 400 μ l dosage are than the performance of 200 μ l dosage better (all standardization).With the SAIB of deposit C at 77: 15: 8: NMP: preparation and to the rat administration in the ethanol carrier.As shown in figure 13, when control peptide dosage, dose volume had no significant effect continuing delivery parameter in initial 3 days; But after 3 days, 400 μ l dosage are better than the performance of 200 μ l dosage.This shows that increasing volume injected can promote to continue to send.

PLA type in the SAIB carrier is to the influence of rat pharmacokinetic property. with the SAIB of precipitate A at 75: 5: 20: PLA: be formulated as 50mg/g in the NMP carrier and to rat administration 400 μ l.As shown in figure 14, the PLA type is changed into 3M from 3L and can be reduced C _Max, increase t _MaxAnd increase t _0.01This shows that the PLA type can influence and continues to send.

The peptide precipitation form is to the influence of rat pharmacokinetic property. and precipitate A, B, E and G are at 75: 5: 20 SAIB: PLA3M: be formulated as 50mg/g in the NMP carrier and to rat administration 400 μ l.As shown in figure 15, in initial precipitation process, increase zinc content and can reduce C _Max, increase t _MaxAnd increase t _0.01(A and B).In low zinc precipitation, add zinc sulfate and can reduce C as lyophilizing salt _Max, increase t _MaxAnd increase t _0.01(A and E).When final precipitation comprises zinc sulfate as lyophilizing salt, replace the not lasting delivery parameter (E and G) of influence of pH precipitation with the zinc precipitation.Even total zinc content is similar, in this carrier, deposit E and G are not so good as deposit B and do very well.This show zinc be incorporated into precipitation mode (for example, how to precipitate or spray) influence significantly peptide continue send.

Polymer type and dose volume in the monkey body to the influence of pharmacokinetic property.Deposit D is at SAIB: PLA: preparation and to the macaque administration in the NMP excipient.As shown in figure 16, excipient changed into 74: 11: 15 from 75: 5: 20 (400 μ l dosage) not appreciable impact continue delivery parameter; But the both is better than the aqueous solution performance.The dose volume that increases by 74: 11: 15 can reduce C to 1000 μ l _Max, reduce t _MaxAnd increase t _0.01This shows that changing dose volume can influence and continue to send.

PLA and PLGA gel systems

Substrate: the solvent ratio is to the influence of rat pharmacokinetic property. and precipitate A and D are respectively at PLGA1A: be formulated as 50mg/g in the NMP carrier and to rat administration 400 μ l.As shown in figure 17, improve substrate: solvent ratio (more PLGA) can reduce C _Max, increase t _MaxAnd increase t _0.01This shows that the amount of polymer in the carrier can influence and continues to send.

Polymer type is to the influence of rat pharmacokinetic property. and precipitate A and D are respectively at polymer: be formulated as 50mg/g in the NMP carrier and to rat administration 400 μ l.As shown in figure 17, improve polymer molecular weight and L: the G ratio can reduce C simultaneously _Max, increase t _MaxAnd increase t _0.01With deposit B at polymer: be formulated as 50mg/g in the NMP carrier and to rat administration 400 μ l.As shown in figure 18, improve L: the G ratio can reduce C _Max, increase t _MaxAnd increase t _0.01Improve polymer molecular weight and can reduce C _Max, increase t _MaxAnd increase t _0.01This shows that polymer type in the carrier can influence and continues to send.

Type of solvent is to the influence of rat pharmacokinetic property. and deposit B is at PLGA1A: be formulated as 50mg/g in the solvent carrier and to rat administration 400 μ l.As shown in figure 18, solvent is changed into glyceryl triacetate from NMP only increases t _0.01This shows that type of solvent in the carrier can influence and continues to send.

Peptide concentration is to the influence of rat pharmacokinetic property. with the PLGA1A of deposit B: preparation and to rat administration 400 μ l in the NMP carrier at 50: 50.As shown in figure 19, increase dosage and can reduce C _Max, increase t _MaxAnd minimizing t _0.01(all standardization).Precipitation H was at 40: 60 PLA3L: preparation and to rat administration 400 μ l in the NMP carrier.As shown in figure 19, increase dosage and can reduce C _Max, increase t _MaxAnd minimizing t _0.01(all standardization).This show PLA and PLGA carrier can provide the high dose peptide continue send.

Peptide form (no zinc) is to the influence of rat pharmacokinetic property. will precipitate I and spray-dried materials at 40: 60 PLA3L: be formulated as 50mg/g in the NMP carrier and to rat administration 400 μ l.As shown in figure 20, change not zinciferous peptide form and do not change lasting delivery parameter.This shows that the not appreciable impact of physical form of zinciferous peptide does not continue to send.

The peptide form is to the influence of rat pharmacokinetic property. will precipitate H, J, K and L at 40: 60 PLA3L: be formulated as 50mg/g in the NMP excipient and to rat administration 400 μ l.As shown in figure 21, washing precipitation reduces zinc content thus, does not change to continue delivery parameter (J and L).The precipitation significantly different really with the sedimentary way of act of unwashed low zinc (K and L) of washing.Methanol from 50: 50: precipitation causes the C that reduces the aqueous solution _MaxWith the t that increases _MaxThis shows in this carrier, does not influence with the amount of the zinc of peptide co-precipitation to continue to send; But the solution appreciable impact that is settled out peptide is sent.

Cation type is to the influence of rat pharmacokinetic property. polycalcium and ferrum are deposited in 40: 60 PLA3L: in the NMP carrier preparation and to rat administration 400 μ l.As shown in figure 22, all preparations have shown that certain continuing send, and wherein iron preparation is better than the calcium preparation performance.This show other precipitated cationic beyond dezincifying can provide peptide continue send.

Polymer type and peptide form are to the influence of the pharmacokinetic property of monkey. be deposited in polymer: in the NMP carrier preparation and to the macaque administration.As shown in figure 23, with 40: 60 PLGA1A: NMP carrier and precipitate A are changed into 40: 60 PLA3L simultaneously: NMP carrier and precipitation J can reduce C _Max, reduce t _MaxAnd increase t _0.01This preparation method that shows polymer property (for example type and molecular weight) and peptide is significant to continuing to send.

Intermediate processing is to the influence of rat pharmacokinetic property. and precipitation H, J, M and N are respectively at 40: 60 PLA3L: preparation and to rat administration 400 μ l in the NMP carrier.As shown in figure 24, standard precipitation J is respectively the analog (promptly having similar peptide and zinc content) of spraying precipitation M and N with H.In each case, spraying precipitation demonstrates the C of increase _MaxAnd t _MaxThe plasma concentration that spraying is deposited in when finishing in a week is greater than or equal to the sedimentary plasma concentration of standard.This shows and standard precipitated phase ratio, and what the spraying precipitation can provide improvement continues to send potentiality.

For illustration purpose, the aforementioned description of particular provided by the invention is described in detail.According to describing and explanation, others skilled in the art can under the situation of basic conception, easily revise and/or adjust described embodiment for multiple use by using existing knowledge; Therefore, these modifications and/or adjustment should be included in the implication and scope of appended claims.

Sequence table

＜110〉Trimeris Inc.

＜120〉be used for the novel formulation of delivery of antiviral peptide therapeutic agent

＜130〉based on 60/921,704 application

<160>56

＜170〉PatentIn is 3.2 editions

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Trp?Asn?Ala?Ser?Trp?Ser?Asn?Lys?Ser?Leu?Glu?Gln?Ile?Trp?Asn?Asn

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Met?Thr?Trp?Met?Glu?Trp?Asp?Arg?Glu?Ile?Asn?Asn?Tyr?Thr?Ser?Leu

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Ile?His?Ser?Leu?Ile?Glu?Glu?Ser?Gln?Asn?Gln?Gln?Glu?Lys?Asn?Glu

35??????????????????40??????????????????45

Gln?Glu?Leu?Leu?Glu?Leu?Asp?Lys?Trp?Ala?Ser?Leu?Trp?Asn?Trp?Phe

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Tyr?Thr?Ser?Leu?Ile?His?Ser?Leu?Ile?Glu?Glu?Ser?Gln?Asn?Gln?Gln

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Glu?Lys?Asn?Glu?Gln?Glu?Leu?Leu?Glu?Leu?Asp?Lys?Trp?Ala?Ser?Leu

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Met?Thr?Trp?Met?Glu?Trp?Asp?Arg?Glu?Ile?Asn?Asn?Tyr?Thr?Ser?Leu

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Ile?His?Ser?Leu?Ile?Glu?Glu?Ser?Gln?Asn?Gln?Gln?Glu?Lys?Asn?Glu

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Gln?Glu?Leu?Leu

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Trp?Met?Glu?Trp?Asp?Arg?Glu?Ile?Asn?Asn?Tyr?Thr?Ser?Leu?Ile?His

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Ser?Leu?Ile?Glu?Glu?Ser?Gln?Asn?Gln?Gln?Glu?Lys?Asn?Glu?Gln?Glu

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Leu?Leu?Glu?Leu

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Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg

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Ile?Glu?Ala?Leu?Ile?Arg?Ala?Ala?Gln?Glu?Gln?Gln?Glu?Lys?Asn?Glu

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Ala?Ala?Leu?Arg?Glu?Leu

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Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg

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Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg

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Ile?Glu?Ala?Leu?Ile?Arg?Ala?Ala?Gln?Glu?Gln?Gln?Glu?Lys?Leu?Glu

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Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg

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Ile?Glu?Ala?Leu?Leu?Arg?Ala?Ala?Gln?Glu?Gln?Gln?Glu?Lys?Asn?Glu

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Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg

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Ile?Glu?Ala?Leu?Leu?Arg?Ala?Leu?Gln?Glu?Gln?Gln?Glu?Lys?Asn?Glu

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Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg

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Ile?Glu?Ala?Leu?Leu?Arg?Ala?Ala?Gln?Glu?Gln?Gln?Glu?Lys?Leu?Glu

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Ala?Ala?Leu?Arg?Glu?Leu

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Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg

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Ile?Glu?Ala?Leu?Ile?Arg?Ala?Leu?Gln?Glu?Gln?Gln?Glu?Lys?Leu?Glu

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Ala?Ala?Leu?Arg?Glu?Leu

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Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg

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Ile?Glu?Ala?Leu?Ile?Arg?Ala?Ile?Gln?Glu?Gln?Gln?Glu?Lys?Leu?Glu

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Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg

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Ile?Glu?Ala?Leu?Ile?Arg?Ala?Leu?Gln?Glu?Gln?Gln?Glu?Lys?Ile?Glu

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Ala?Ala?Leu?Arg?Glu?Leu

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Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg

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Ile?Glu?Ala?Leu?Leu?Arg?Ala?Ile?Gln?Glu?Gln?Gln?Glu?Lys?Asn?Glu

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<222>(6)..(6)

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<222>(17)..(17)

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<222>(20)..(21)

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<222>(24)..(24)

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<222>(25)..(25)

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<222>(27)..(28)

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Xaa?Xaa?Xaa?Glu?Ala?Xaa?Asp?Arg?Ala?Xaa?Ala?Glu?Xaa?Ala?Ala?Arg

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Xaa?Glu?Ala?Xaa?Xaa?Arg?Ala?Xaa?Xaa?Glu?Xaa?Xaa?Glu?Lys?Xaa?Glu

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Ala?Ala?Xaa?Arg?Glu?Xaa

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Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Leu?Gln?Glu

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Gln?Gln?Glu?Lys?Asn?Glu?Ala?Ala?Leu?Arg?Glu

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Gln?Gln?Glu?Lys?Asn?Glu?Ala?Ala?Leu?Arg?Glu?Leu

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Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile?Ala

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Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Leu?Gln?Glu

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Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile

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Ala?Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Leu?Gln?Glu

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<400>25

Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala

1???????????????5

<210>26

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>26

Ile?Ala?Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Leu?Gln

1???????????????5???????????????????10??????????????????15

Glu

<210>27

<211>8

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>27

Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg

1???????????????5

<210>28

<211>18

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>28

Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Leu

1???????????????5???????????????????10??????????????????15

Gln?Glu

<210>29

<211>20

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>29

Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg

1???????????????5???????????????????10??????????????????15

Ile?Glu?Ala?Leu

20

<210>30

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>30

Leu?Arg?Ala?Leu?Gln?Glu?Gln?Gln?Glu?Lys?Asn?Glu?Ala?Ala?Leu?Arg

1???????????????5???????????????????10??????????????????15

Glu

<210>31

<211>18

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>31

Leu?Arg?Ala?Leu?Gln?Glu?Gln?Gln?Glu?Lys?Asn?Glu?Ala?Ala?Leu?Arg

1???????????????5???????????????????10??????????????????15

Glu?Leu

<210>32

<211>18

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>32

Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg

1???????????????5???????????????????10??????????????????15

Ile?Glu

<210>33

<211>19

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>33

Ala?Leu?Leu?Arg?Ala?Leu?Gln?Glu?Gln?Gln?Glu?Lys?Asn?Glu?Ala?Ala

1???????????????5???????????????????10??????????????????15

Leu?Arg?Glu

<210>34

<211>20

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>34

Ala?Leu?Leu?Arg?Ala?Leu?Gln?Glu?Gln?Gln?Glu?Lys?Asn?Glu?Ala?Ala

1???????????????5???????????????????10??????????????????15

Leu?Arg?Glu?Leu

20

<210>35

<211>26

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>35

Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Leu?Gln?Glu?Gln?Gln

1???????????????5???????????????????10??????????????????15

Glu?Lys?Asn?Glu?Ala?Ala?Leu?Arg?Glu?Leu

20??????????????????25

<210>36

<211>27

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>36

Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Leu?Gln?Glu?Gln

1???????????????5???????????????????10??????????????????15

Gln?Glu?Lys?Asn?Glu?Ala?Ala?Leu?Arg?Glu?Leu

20??????????????????25

<210>37

<211>28

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>37

Ala?Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Leu?Gln?Glu

1???????????????5???????????????????10??????????????????15

Gln?Gln?Glu?Lys?Asn?Glu?Ala?Ala?Leu?Arg?Glu?Leu

20??????????????????25

<210>38

<211>29

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>38

Ile?Ala?Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Leu?Gln

1???????????????5???????????????????10??????????????????15

Glu?Gln?Gln?Glu?Lys?Asn?Glu?Ala?Ala?Leu?Arg?Glu?Leu

20??????????????????25

<210>39

<211>30

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>39

Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Leu

1???????????????5???????????????????10??????????????????15

Gln?Glu?Gln?Gln?Glu?Lys?Asn?Glu?Ala?Ala?Leu?Arg?Glu?Leu

20??????????????????25??????????????????30

<210>40

<211>26

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>40

Thr?Thr?Trp?Glu?Ala?Trp?Asp?Arg?Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg

1???????????????5???????????????????10??????????????????15

Ile?Glu?Ala?Leu?Leu?Arg?Ala?Leu?Gln?Glu

20??????????????????25

<210>41

<211>14

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>41

Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Ala?Gln?Glu

1???????????????5???????????????????10

<210>42

<211>11

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>42

Gln?Gln?Glu?Lys?Leu?Glu?Ala?Ala?Leu?Arg?Glu

1???????????????5???????????????????10

<210>43

<211>12

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>43

Gln?Gln?Glu?Lys?Leu?Glu?Ala?Ala?Leu?Arg?Glu?Leu

1???????????????5???????????????????10

<210>44

<211>15

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>44

Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Ala?Gln?Glu

1???????????????5???????????????????10??????????????????15

<210>45

<211>16

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>45

Ala?Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Ala?Gln?Glu

1???????????????5???????????????????10??????????????????15

<210>46

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>46

Ile?Ala?Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Ala?Gln

1???????????????5???????????????????10??????????????????15

Glu

<210>47

<211>18

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>47

Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Ala

1???????????????5???????????????????10??????????????????15

Gln?Glu

<210>48

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>48

Leu?Arg?Ala?Ala?Gln?Glu?Gln?Gln?Glu?Lys?Leu?Glu?Ala?Ala?Leu?Arg

1???????????????5???????????????????10??????????????????15

Glu

<210>49

<211>19

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>49

Leu?Arg?Ala?Ala?Leu?Gln?Glu?Gln?Gln?Glu?Lys?Leu?Glu?Ala?Ala?Leu

1???????????????5???????????????????10??????????????????15

Arg?Glu?Leu

<210>50

<211>19

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>50

Ala?Leu?Leu?Arg?Ala?Ala?Gln?Glu?Gln?Gln?Glu?Lys?Leu?Glu?Ala?Ala

1???????????????5???????????????????10??????????????????15

Leu?Arg?Glu

<210>51

<211>20

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>51

Ala?Leu?Leu?Arg?Ala?Ala?Gln?Glu?Gln?Gln?Glu?Lys?Leu?Glu?Ala?Ala

1???????????????5???????????????????10??????????????????15

Leu?Arg?Glu?Leu

20

<210>52

<211>26

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>52

Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Ala?Gln?Glu?Gln?Gln

1???????????????5???????????????????10??????????????????15

Glu?Lys?Leu?Glu?Ala?Ala?Leu?Arg?Glu?Leu

20??????????????????25

<210>53

<211>27

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>53

Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Ala?Gln?Glu?Gln

1???????????????5???????????????????10??????????????????15

Gln?Glu?Lys?Leu?Glu?Ala?Ala?Leu?Arg?Glu?Leu

20??????????????????25

<210>54

<211>28

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>54

Ala?Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Ala?Gln?Glu

1???????????????5???????????????????10??????????????????15

Gln?Gln?Glu?Lys?Leu?Glu?Ala?Ala?Leu?Arg?Glu?Leu

20??????????????????25

<210>55

<211>29

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>55

Ile?Ala?Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Ala?Gln

1???????????????5???????????????????10??????????????????15

Glu?Gln?Gln?Glu?Lys?Leu?Glu?Ala?Ala?Leu?Arg?Glu?Leu

20??????????????????25

<210>56

<211>30

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic

<400>56

Ala?Ile?Ala?Glu?Tyr?Ala?Ala?Arg?Ile?Glu?Ala?Leu?Leu?Arg?Ala?Ala

1???????????????5??????????????????10??????????????????15

Gln?Glu?Gln?Gln?Glu?Lys?Leu?Glu?Ala?Ala?Leu?Arg?Glu?Leu

20??????????????????25??????????????????30

Claims (49)

1. the compositions that comprises solvent, Binder Materials and bioactive molecule, wherein after being applied to the patient, described compositions forms substrate and is using the C that the described bioactive molecule of at least 10 μ g/ml was provided in 12 hours _Max, the blood plasma level with at least 1 μ g/ml continues to discharge at least 7 days then.

2. the compositions that comprises solvent, Binder Materials and peptide, wherein said peptide is selected from T20, T1249, T897, T2635, T999 and T1144, or the combination of above-mentioned peptide.

3. compositions as claimed in claim 2, wherein said solvent are NMP.

4. compositions as claimed in claim 2, wherein said Binder Materials are SAIB.

5. compositions as claimed in claim 2, wherein said Binder Materials are PLA, PLG, PLGA or PLGA-glucose.

6. compositions as claimed in claim 2, the amount of wherein said Binder Materials are 30% to 85% weight.

7. compositions as claimed in claim 2, the amount of wherein said Binder Materials are 30% to 80% weight.

8. compositions as claimed in claim 2, the amount of wherein said Binder Materials are 30% to 70% weight.

9. compositions as claimed in claim 2, the amount of wherein said Binder Materials are 60% to 85% weight.

10. compositions as claimed in claim 2, the amount of wherein said Binder Materials are 65% to 85% weight.

11. compositions as claimed in claim 2, the amount of wherein said Binder Materials are 75% to 85% weight.

12. compositions as claimed in claim 2, wherein said peptide are T1144.

13. compositions as claimed in claim 2, wherein said compositions is being used the C that the described bioactive molecule of at least 10 μ g/ml was provided in 12 hours _Max, the blood plasma level with at least 1 μ g/ml continues to discharge at least 7 days then.

14. compositions as claimed in claim 2, wherein said compositions further comprise at least a other bioactive molecule.

15. compositions as claimed in claim 14, wherein said other bioactive molecule is an antiviral agent.

16. compositions as claimed in claim 15, wherein said antiviral agent are peptide.

17. compositions as claimed in claim 15, wherein said antiviral agent are cytokine.

18. compositions as claimed in claim 15, wherein said antiviral agent are reverse transcriptase inhibitors.

19. being virus mRNA, compositions as claimed in claim 15, wherein said antiviral agent add the medicated cap inhibitor.

20. compositions as claimed in claim 2, wherein said Binder Materials are two or more mixtures of material that are selected from PLA, PLG, PLGA or PLGA-glucose.

21. compositions as claimed in claim 2, wherein said peptide is dissolved in described solvent and the Binder Materials.

22. compositions as claimed in claim 2, wherein said peptide is suspended in described solvent and the Binder Materials.

23. compositions as claimed in claim 22, the peptide of wherein said suspension are spray-dried forms.

24. compositions as claimed in claim 23, the peptide of wherein said suspension is the saliniferous spray-dried forms of bag.

25. compositions as claimed in claim 24, the peptide of wherein said suspension is the zinciferous spray-dried forms of bag.

26. compositions as claimed in claim 24, the peptide of wherein said suspension is the calcareous spray-dried forms of bag.

27. compositions as claimed in claim 22, the peptide of wherein said suspension are precipitation form.

28. compositions as claimed in claim 27, the peptide of wherein said suspension is the saliniferous precipitation form of bag.

29. compositions as claimed in claim 28, the peptide of wherein said suspension is the zinciferous precipitation form of bag.

30. compositions as claimed in claim 28, the peptide of wherein said suspension is the calcareous precipitation form of bag.

31. compositions as claimed in claim 28, the peptide of wherein said suspension is the ferruginous precipitation form of bag.

32. continue the method for release peptide in patient's body, wherein said method comprises to the patient uses the compositions that comprises solvent, Binder Materials and peptide, wherein said peptide is selected from T20, T1249, T897, T2635, T999 and T1144, or the combination of above-mentioned peptide.

33. method as claimed in claim 32, wherein said compositions is used by subcutaneous injection.

34. method as claimed in claim 32, wherein said solvent are NMP.

35. method as claimed in claim 32, wherein said Binder Materials are SAIB.

36. method as claimed in claim 32, wherein said Binder Materials are PLA, PLG, PLGA or PLGA-glucose.

37. method as claimed in claim 32, wherein said peptide are T1144.

Infect related indication method 38. improve HIV, wherein said method comprises to the HIV infected patient uses the compositions that comprises solvent, Binder Materials and peptide, wherein said peptide is selected from T20, T1249, T897, T2635, T999 and T1144, or the combination of above-mentioned peptide.

39. method as claimed in claim 38, wherein said compositions is used by subcutaneous injection.

40. method as claimed in claim 38, wherein said solvent are NMP.

41. method as claimed in claim 38, wherein said Binder Materials are SAIB.

42. method as claimed in claim 38, wherein said Binder Materials are PLA, PLG, PLGA or PLGA-glucose.

43. method as claimed in claim 38, wherein said peptide are T1144.

44. method as claimed in claim 38, wherein said compositions further comprise at least a other bioactive molecule.

45. method as claimed in claim 44, wherein said other bioactive molecule is an antiviral agent.

46. method as claimed in claim 44, wherein said antiviral agent are peptide.

47. method as claimed in claim 44, wherein said antiviral agent are cytokine.

48. method as claimed in claim 44, wherein said antiviral agent are reverse transcriptase inhibitors.

49. being virus mRNA, method as claimed in claim 44, wherein said antiviral agent add the medicated cap inhibitor.

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